Microfluidic systems including three-dimensionally arrayed channel networks

ABSTRACT

The present invention provides, in certain embodiments, improved microfluidic systems and methods for fabricating improved microfluidic systems, which contain one or more levels of microfluidic channels. The inventive methods can provide a convenient route to topologically complex and improved microfluidic systems. The microfluidic systems provided according to the invention can include three-dimensionally arrayed networks of fluid flow paths therein including channels that cross over or under other channels of the network without physical intersection at the points of cross over. The microfluidic networks of the invention can be fabricated via replica molding processes, also provided by the invention, utilizing mold masters including surfaces having topological features formed by photolithography. The microfluidic networks of the invention are, in some cases, comprised of a single replica molded layer, and, in other cases, are comprised of two, three, or more replica molded layers that have been assembled to form the overall microfluidic network structure. The present invention also describes various novel applications for using the microfluidic network structures provided by the invention.

RELATED APPLICATIONS

[0001] This application is a continuation of PCT InternationalApplication No. PCT/US01/16973 filed May 25, 2001, which was publishedunder PCT Article 21(2) in English, and claims priority viaPCT/US01/16973 to U.S. application Ser. No. 09/578,589, filed May 25,2000. Both applications are hereby incorporated by reference.

FIELD OF INVENTION

[0002] The present invention involves microfluidic network structures,methods for fabricating microfluidic network structures, and methods forusing such structures.

BACKGROUND OF THE INVENTION

[0003] The need for complexity in microfluidic systems is increasingrapidly as sophisticated functions—chemical reactions and analyses,bioassays, high-throughput screens, and sensors—are being integratedinto single microfluidic devices. Complex systems of channels requiremore complex connectivity than can be generated in conventionaltwo-dimensional microfluidic systems having a single level of channels,since such typical single-level designs do not allow two channels tocross without fluidically connecting. Most methods for fabricatingmicrofluidic channels are based on photolithographic procedures, andyield such two-dimensional systems. There are a number of morespecialized procedures, such as stereolithography (see for example, K.Ikuta, K. Hirowatari, T. Ogata, Proc. IEEE MEMS '94, Oiso, Japan, Jan.25-28, 1994, pp. 1-6), laser-chemical three-dimensional writing (see forexample, T. M. Bloomstein, D. J. Ehrlich, J. Vac. Sci. Technol. B, Vol.10, pp. 2671-2674, 1992), and modular assembly (see for example, C.Gonzalez, R. L. Smith, D. G. Howitt, S. D. Collins, Sens. Actuators A,Vol. 66, pp. 315-332, 1998), that yield three-dimensional structures,but these methods are typically time consuming, difficult to perform,and expensive, and are thus not well suited for either prototyping ormanufacturing, and are also not capable of making certain types ofstructures. Better methods for generating complex three-dimensionalmicrofluidic systems are needed to accelerate the development ofmicrofluidic technology. The present invention, in some embodiments,provides such improved methods for generating complex three-dimensionalmicrofluidic systems.

[0004] It is known to use a stamp or mold to transfer patterns to asurface of a substrate (see for example, R. S. Kane, S. Takayama, E.Ostuni, D. E. Ingber, G. M. Whitesides, Biomaterials, Vol. 20, pp.2363-2376, 1999; and Y. Xia, G. M. Whitesides, Angew. Chem. Int. Ed.Engl., Vol. 37, pp. 551-575, 1998; U.S. Pat. No. 5,512,131;International Pat. Publication No. WO 97/33737, published Sep. 18,1997). Most conventional soft lithographic techniques, for example,microcontact printing (μCP) (see for example, C. S. Chen, M. Mrksich, S.Huang, G. M. Whitesides, D. E. Ingber, Science, Vol. 276, pp. 1425-1428,1997; A. Bernard, E. Delamarche, H. Schmid, B. Michel, H. R. Bosshard,H. Biebuyck, Langmuir, Vol. 14, pp. 2225-2229, 1998) and micromolding incapillaries (MIMIC) (see for example, N. L. Jeon, I. S. Choi, B. Xu, G.M. Whitesides, Adv. Mat., Vol. 11, pp. 946-949, 1999; E. Delamarche, A.Bernard, H. Schmid, B Michel, H. Biebuyck, Science, Vol. 276, pp.779-781, 1997; E. Delamarche, A. Bernard, H. Schmid, A. Bietsch, B.Michel, h. Biebuyck, J. Am. Chem. Soc., Vol. 120, pp. 500-508, 1998; A.Folch, A. Ayon, O. Hurtado, M. A. Schmidt, M. Toner, J. Biomech. Eng.,Vol. 121, pp. 28-34, 1999; A. Folch, M. Toner, Biotech. Prog., Vol. 14,pp. 388-392, 1998), have been limited to procedures that pattern onesubstance at a time, or to relatively simple, continuous patterns. Theseconstraints are both topological and practical. The surface of a stampin μCP, or of a channel system in MIMIC, is effectively atwo-dimensional structure. In μCP, this two-dimensionality of the stamplimits the types of patterns that can be transferred to those comprisinga single “color” of ink in the absence of a way of selectively “inking”different regions of the stamp with different materials. Patterning ofmultiple “inks” using conventional methods requires multiple steps ofregistration and stamping. In MIMIC, the two-dimensional channel systemlimits patterning to relatively simple, continuous structures orrequires multiple patterning steps.

[0005] There remains a general need in the art for improved methods forforming patterns on surfaces with soft lithographic techniques, and forproviding techniques able to pattern onto a surface arbitrarytwo-dimensional patterns and able to form complex patterns comprised ofmultiple regions, where different regions of the pattern can comprisedifferent materials, on a surface without the need for multiple steps ofregistration or stamping and without the need to selectively “ink”different regions of the stamp with different materials. The presentinvention, in some embodiments, provides such improved methods forforming patterns on surfaces with soft lithographic techniques.

SUMMARY OF THE INVENTION

[0006] The present invention involves, in certain embodiments, improvedmicrofluidic systems and procedures for fabricating improvedmicrofluidic systems, which contain one or more levels of microfluidicchannels. The inventive methods can provide a convenient route totopologically complex and improved microfluidic systems. The presentinvention also, in some embodiments, involves microfluidic systems andmethods for fabricating complex patterns of materials, such asbiological materials and cells, on surfaces. In such embodiments, theinvention involves microfluidic surface patterning systems and methodsfor fabricating complex, discontinuous patterns on surfaces that canincorporate or deposit multiple materials onto a surface. The presentinvention, in some embodiments, can provide improved stamps formicrocontact surface patterning able to pattern onto a surface arbitrarytwo-dimensional patterns and able to pattern multiple substances onto asurface without the need for multiple steps of registration or stampingduring patterning and without the need to selectively “ink” differentregions of the stamp with different materials.

[0007] According to one embodiment of the invention, a microfluidicnetwork is disclosed. The microfluidic network comprises a polymericstructure including therein at least a first and a secondnon-fluidically interconnected fluid flow paths. At least the first flowpath comprises a series of interconnected channels within the polymericstructure. The series of interconnected channels includes at least onefirst channel disposed within a first level of the structure, at leastone second channel disposed within a second level of the structure, andat least one connecting channel fluidically interconnecting the firstchannel and the second channel. At least one channel within thestructure has a cross-sectional dimension not exceeding about 500 μm.The structure includes at least one channel disposed within the firstlevel of the structure that is non-parallel to at least one channeldisposed within the second level of the structure.

[0008] In another embodiment of the invention, a microfluidic network isdisclosed. The microfluidic network comprises an elastomeric structureincluding therein at least one fluid flow path. The flow path comprisesa series of interconnected channels within the structure. The series ofinterconnected channels includes at least one first channel disposedwithin a first level of the structure, at least one second channeldisposed within a second level of the structure, and at least oneconnecting channel fluidically interconnecting the first channel and thesecond channel. At least one channel within the structure has across-sectional dimension not exceeding about 500 μm, and the structureincludes at least one channel disposed within the first level of thestructure that is non-parallel to at least one channel disposed withinthe second level of the structure.

[0009] In yet another embodiment, a polymeric membrane is disclosed. Thepolymeric membrane comprises a first surface including at least onechannel disposed therein, a second surface including at least onechannel disposed therein, and a polymeric region intermediate the firstsurface and the second surface. The intermediate region includes atleast one connecting channel therethrough fluidically interconnectingthe channel disposed in the first surface and the channel disposed inthe second surface of the membrane. At least one channel has across-sectional dimension not exceeding about 500 μm.

[0010] In another embodiment of the invention, a method for forming amicrofluidic network structure is disclosed. The method comprisesproviding at least one mold substrate, forming at least one topologicalfeature on a surface of the mold substrate to form a first mold master,contacting the surface with a first hardenable liquid, hardening theliquid thereby creating a first molded replica of the surface, removingthe first molded replica from the first mold master, and assembling thefirst molded replica into a structure comprising a microfluidic network.The assembled microfluidic network structure has at least one fluid flowpath comprising a series of interconnected channels within thestructure. The series of interconnected channels includes at least onefirst channel disposed within a first level of the structure, at leastone second channel disposed within a second level of the structure, andat least one connecting channel fluidically interconnecting the firstchannel and the second channel. At least one of the channels within thestructure has a cross-sectional dimension not exceeding about 500 μm.The structure includes at least one channel disposed within the firstlevel of the structure that is non-parallel to at least one channeldisposed within the second level of the structure.

[0011] In yet another embodiment, a method for forming a moldedstructure is disclosed. The method comprises providing at least one moldsubstrate and forming at least one two-level topological feature havingat least one lateral dimension not exceeding 500 μm on a surface of thesubstrate to form a mold master. The two-level topological feature ischaracterized by a first portion having a first depth or height withrespect to a region of the surface adjacent to the feature, and a secondportion integrally connected with the first portion having a seconddepth or height with respect to the region of the surface adjacent tothe feature that is greater than the first depth or height. The methodfurther comprises contacting the surface with a hardenable liquid,hardening the liquid thereby creating a molded replica of the surface,and removing the molded replica from the mold master.

[0012] In another embodiment of the invention, a method for formingtopological features on a surface of a material is disclosed. The methodcomprises exposing portions of a surface of a first layer of photoresistto radiation in a first pattern, coating the surface of the first layerof photoresist with a second layer of photoresist, exposing portions ofa surface of the second layer of photoresist to radiation in a secondpattern different from the first pattern, and developing the first andsecond photoresist layers with a developing agent. The developing stepyields a positive relief pattern in photoresist that includes at leastone two-level topological feature having at least one cross-sectionaldimension not exceeding 500 μm. The two-level topological feature ischaracterized by a first portion having a first height with respect tothe surface of the material and a second portion, integrally connectedto the first portion, having a second height with respect to the surfaceof the material.

[0013] In yet another embodiment, a method for forming a moldedstructure is disclosed. The method involves providing a first moldmaster having a surface formed of an elastomeric material and includingat least one topological feature with at least one cross-sectionaldimension not exceeding about 500 μm thereon. The method furthercomprises providing a second mold master having a surface including atleast one topological feature with at least one cross-sectionaldimension not exceeding about 500 μm thereon. The method furthercomprises placing a hardenable liquid in contact with the surface of atleast one of the first and second mold master, bringing the surface ofthe first mold master into at least partial contact with the surface ofthe second mold master, hardening the liquid thereby creating a moldedreplica of the surface of the first mold master and the surface of thesecond mold master, and removing the molded replica from at least one ofthe mold masters.

[0014] In another embodiment of the invention, a method for forming amolded structure is disclosed. The method involves providing a firstmold master having a surface including at least a first topologicalfeature with at least one cross-sectional dimension not exceeding about500 μm thereon and at least a second topological feature comprising afirst alignment element. The method further comprises providing a secondmold master having a surface including at least a first topologicalfeature with at least one cross-sectional dimension not exceeding about500 μm thereon and at least a second topological feature comprising asecond alignment element having a shape that is mateable to the shape ofthe first alignment element. The method further comprises placing ahardenable liquid in contact with the surface of at least one of thefirst and second mold master, bringing the surface of the first moldmaster into at least partial contact with the surface of the second moldmaster, aligning the first topological features of the first and secondmold masters with respect to each other by adjusting a position of thefirst mold master with respect to a position of the second mold masteruntil the first alignment element matingly engages and interdigitateswith the second alignment element, hardening the liquid thereby creatinga molded replica of the surface of the first mold master and the surfaceof the second mold master, and removing the molded replica from at leastone of the mold masters.

[0015] In yet another embodiment of the invention, a method for aligningand sealing together surfaces is disclosed. The method comprisesdisposing two surfaces, at least one of which is oxidized, adjacent toeach other such that they are separated from each other by a continuouslayer of a liquid that is essentially non-reactive with the surfaces,aligning the surfaces with respect to each other, and removing theliquid from between the surfaces, thereby sealing the surfaces togethervia a chemical reaction between the surfaces.

[0016] In another embodiment of the invention, a method for molding anarticle is disclosed. The method comprises providing a first mold masterhaving a surface with a first set of surface properties and providing asecond mold master having a surface with a second set of surfaceproperties. At least one of the first and second mold masters has asurface including at least one topological feature with at least onecross-sectional dimension not exceeding about 500 μm thereon. The methodfurther comprises placing a hardenable liquid in contact with thesurface of at least one of the first and second mold masters, bringingthe surface of the first mold master into at least partial contact withthe surface of the second mold master, hardening the liquid therebycreating a molded replica of the surface of the first mold master andthe surface of the second mold master, separating the mold masters fromeach other, and removing the molded replica from the surface of thefirst mold master while leaving the molded replica in contact with andsupported by the surface of the second mold master.

[0017] In yet another embodiment, a microfluidic network is disclosed.The microfluidic network comprises a polymeric structure includingtherein at least a first and a second non-fluidically interconnectedfluid flow paths. The first flow path comprises at least twonon-colinear interconnected channels disposed within a first plane, andthe second flow path comprises at least one channel disposed within asecond plane that is non-parallel with the first plane. At least onechannel within the structure has a cross-sectional dimension notexceeding about 500 μm.

[0018] In another embodiment of the invention, a microfluidic network isdisclosed. The microfluidic network comprises a polymeric structureincluding therein at least one fluid flow path. The fluid flow path isformed of at least one channel and has a longitudinal axis defined bythe direction of bulk fluid flow within the flow path. The longitudinalaxis of the flow path is not disposed within any single plane.

[0019] In another embodiment of the invention, a method of patterning amaterial surface is disclosed. The method comprises providing a stamphaving a structure including at least one flow path comprising a seriesof interconnected channels within the structure. The series ofinterconnected channels includes at least one first channel disposedwithin an interior region of the structure, at least one second channeldisposed within a stamping surface of the structure defining a firstpattern therein, and at least one connecting channel fluidicallyinterconnecting the first channel and the second channel. The methodfurther comprises contacting the stamping surface with a portion of thematerial surface, and, while maintaining the stamping surface in contactwith the portion of the material surface, at least partially filling theflow path with a fluid so that at least a portion of the fluid contactsthe material surface.

[0020] In yet another embodiment, a method of patterning a materialsurface is disclosed. The method comprises providing a stamp having astructure including at least two non-fluidically interconnected flowpaths therein including a first fluid flow path defining a first patternof channels disposed within a stamping surface of the structure and asecond fluid flow path defining a second pattern of channels disposedwithin the stamping surface of the structure. Each of the first andsecond patterns of channels is non-continuous, and the channels definingthe first pattern are non-intersecting with the channels defining thesecond pattern. The method further comprises contacting the stampingsurface with a portion of the material surface, while maintaining thestamping surface in contact with the portion of the material surface, atleast partially filling the first flow path with a first fluid so thatat least a portion of the first fluid contacts the material surface andat least partially filling the second flow path with a second fluid sothat at least a portion of the second fluid contacts the materialsurface, and removing the stamping surface to provide a pattern on thematerial surface according to the first pattern, which is formed bycontact of the material surface with the first fluid, and according tothe second pattern, which is formed by contact of the material surfacewith the second fluid.

[0021] In another embodiment, a method of patterning a material surfaceis disclosed, the method involves providing a stamp having a structureincluding at least one non-linear fluid flow path therein in fluidcommunication with a stamping surface of the structure. The methodfurther involves contacting the stamping surface with a portion of thematerial surface and, while maintaining the stamping surface in contactwith the portion of the material surface, at least partially filling theflow path with a fluid so that at least a portion of the fluid contactsthe material surface.

[0022] Other advantages, novel features, and objects of the inventionwill become apparent from the following detailed description of theinvention when considered in conjunction with the accompanying drawings,which are schematic and which are not intended to be drawn to scale. Inthe figures, each identical or nearly identical component that isillustrated in various figures is represented by a single numeral. Forpurposes of clarity, not every component is labeled in every figure, noris every component of each embodiment of the invention shown whereillustration is not necessary to allow those of ordinary skill in theart to understand the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0023]FIG. 1a is a perspective view of a schematic illustration of amicrofluidic network structure having a series of interconnectedchannels arranged in a “basketweave” configuration;

[0024]FIG. 1b is a two-dimensional projection of the microfluidicnetwork structure of FIG. 1a;

[0025]FIG. 2a is a perspective view of a schematic illustration of asecond embodiment of a microfluidic network structure;

[0026]FIG. 2b is a two-dimensional projection of the microfluidicnetwork structure of FIG. 2a;

[0027]FIG. 3a is a perspective view of a schematic illustration of athird embodiment of a microfluidic network structure;

[0028]FIG. 3b is a two-dimensional projection of the microfluidicnetwork structure of FIG. 3a;

[0029]FIG. 4a is a perspective view of a schematic illustration of afive-level microfluidic network comprising a centrally disposed straightchannel surrounded by a coiled fluid flow path;

[0030]FIG. 4b is a two-dimensional projection of the microfluidicnetwork structure of FIG. 4a;

[0031]FIGS. 5a-5 c are schematic illustrations of one embodiment of thefabrication method for forming a microfluidic network structureaccording to one embodiment of the invention;

[0032]FIGS. 6a-6 c are schematic illustrations of one embodiment of aself-aligning method provided by the invention;

[0033]FIG. 6d is a schematic illustration of a replica molded layer of amicrofluidic network having a perimetric shape for use in one embodimentof a self-aligning method according to the invention;

[0034]FIG. 7 is a schematic illustration of a second embodiment of amicrofluidic network fabrication method according to the invention;

[0035]FIG. 8 is a schematic illustration of a method for forming atwo-level topological feature on a surface of the substrate byphotolithography provided according to the invention;

[0036]FIGS. 9a-9 b are schematic illustrations of a third embodiment forforming a microfluidic network structure according to the invention;

[0037]FIG. 9c is a series of schematic, cross-sectional illustrations ofa modification of the third embodiment for forming the microfluidicnetwork structure of FIGS. 9a-9 b.

[0038]FIG. 10 is a schematic illustration of a method for forming afive-level microfluidic network structure including a straight channelsurrounded by a coiled series of interconnected channels;

[0039]FIG. 11 is a schematic illustration of a pattern on a materialsurface formed with a microfluidic stamp provided according to theinvention;

[0040]FIG. 12a is a perspective view of a schematic illustration of alower and an upper mold master for forming a basketweave microfluidicnetwork structure provided by the invention;

[0041]FIGS. 12b-12 c provide photocopies of photomicrographs of amicrofluidic network characterized by a network of channels arranged ina basketweave configuration in accordance with one embodiment of thepresent invention;

[0042]FIG. 12d is a photocopy of an SEM image of a micromolded structureproduced according to one embodiment of the invention;

[0043]FIG. 13 is a photocopy of a photomicrograph of a microfluidicnetwork comprising a straight channel surrounded by a coiled fluid flowpath comprising a series of interconnected channels, according to oneembodiment of the invention;

[0044]FIG. 14a is a schematic illustration of a microfluidic stampingprocess according to one embodiment of the invention;

[0045]FIG. 14b is a schematic illustration of the fluid flow path layoutof the microfluidic stamp illustrated in FIG. 14a;

[0046]FIG. 14c is a photocopy of a photomicrograph of a patternedsurface produced using the microfluidic stamp illustrated in FIG. 14a;

[0047]FIG. 15a is a schematic illustration of the layout of fluid flowpaths in one embodiment of a microfluidic stamp provided according tothe invention;

[0048]FIG. 15b is a photocopy of photomicrograph of a stamped pattern ona material surface produced using a microfluidic stamp having themicrofluidic network structure illustrated in FIG. 15a;

[0049]FIG. 16a is a schematic illustration of the layout of fluid flowpaths in one embodiment of a microfluidic stamp provided according tothe invention;

[0050]FIGS. 16b-16 d are photocopies of photomicrographs of patternedcells on a material surface deposited using a microfluidic stamp havingthe microfluidic network configuration illustrated in FIG. 16a;

[0051]FIG. 17a is a schematic illustration of the layout of fluid flowpaths in one embodiment of a microfluidic stamp provided according tothe invention; and

[0052]FIGS. 17b-17 e are photocopies of photomicrographs of patternedcells on a material surface deposited using a microfluidic stamp havingthe microfluidic network configuration illustrated in FIG. 17a.

DETAILED DESCRIPTION

[0053] The present invention is directed to fabrication methods forproducing three-dimensional microfluidic network structures, polymericmicrofluidic network structures having a three-dimensional array ofchannels included therein, and various uses for the microfluidicnetworks, for example as a template for forming and depositing complexpatterns on substrates. A “three-dimensional microfluidic network,”“three-dimensional microfluidic network structure,” or“three-dimensional microfluidic stamp” as used herein refers to astructure capable of containing a fluid and/or providing fluid flowtherethrough, which includes at least three channels therein, and maycontain many more; furthermore, the structure includes at least threechannels that are arranged with respect to each other such that thereexists no plane, or curved planar surface, which contains disposedtherein the longitudinal axes of the three channels. The microfluidicnetworks provided according to the invention, because of theirthree-dimensionality of structure, are able, for example, to providechannels within the structure having longitudinal axes (defined as theaxial centerline of the channel aligned parallel to the direction ofbulk fluid flow within the channel) aligned along each of the x, y, andz directional components of space. The ability to produce microfluidicstructures having channels arranged in a three-dimensional networkenables the systems provided according to the invention to includetherein a plurality of channels providing one or more independent fluidflow paths, where the channels and flow paths can be arrayed inarbitrarily complex geometric networks since the channels of thestructures have the capability of crossing over and/or under each otherwithin the structure.

[0054] One way to analogize the capabilities of the microfluidicnetworks, and methods for producing the microfluidic networks, accordingto the invention, is to compare the channel systems of the microfluidicnetworks to a knot in three-dimensional space. The microfluidic networksprovided according to the invention have the ability to fabricate thephysical realization of knots, and thus can include channel systems ofarbitrary topological complexity. In mathematical terms, a knot is aclosed, non-intersecting, curved line in three dimensions. Knots aretypically described in mathematics in terms by their projections onto aplane. For non trivial knots, these projections contain “double points”,which are points where the projected curve crosses itself. A knot canalways be slightly perturbed in three dimensions so that, in projection,it has no triple or higher order points: that is, points where theprojected curve crosses itself three or more times. Hence, knots can bedescribed completely by giving such a two-dimensional projection,together with information about which piece of the curve crosses over orunder the another piece at each double point.

[0055] The microfluidic networks provided according to the invention,because of their three-dimensional channel network structure, are ableto provide a physical realization of the above-mentioned double point.In other words, the structures enable one channel, comprising a flowpath or a segment of a flow path, to cross over or under another channelproviding another flow path, a segment of another flow path, orproviding another segment of the same flow path. Thus, the inventivemicrofluidic networks can provide a physical realization of essentiallyany topological knot system. Likewise, the inventive networks canprovide a physical realization of essentially any arrangement ofinterlinked knots and of arbitrarily complex three-dimensional networksof interconnected channels whose projections onto a plane or surface, asexplained in more detail below, can contain any arbitrary number ofcrossings. As shown and explained in more detail below, in order for theinventive microfluidic networks to avoid intersection of channels attheir points of crossing in the planar projection, there typically areprovided at least three identifiable “levels” within the structure: a“lower” level that contains a channel disposed therein that crosses“under” an “upper” level that contains disposed therein a channel thatcrosses “over” the channel contained in the bottom level, and anintermediate level that isolates the channels of the lower and upperlevels and contains connecting channels penetrating therethrough thatfluidically connect the channels in the lower level and the channels onthe upper level in order to form a fluid flow path comprised of a seriesof interconnected channels. It should be understood that the terms“lower” and “upper” in the present context are intended to suggest onlythe relative positions of the various levels of the structure and arenot meant to imply any particular orientation of the structure in space.For example the structure can be flipped, rotated in space, etc. so thatthe “lower” level is positioned above the “upper” level or the levelscan be positioned side by side, etc. In yet other embodiments involvingflexible structures, the structure can be twisted or bent therebydeforming planar levels into curved surfaces in space such that the“upper” and “lower” levels of the structure may be positioneddifferently with respect to each other at different locations in theoverall structure. In order to produce microfluidic networks witharbitrarily complex channel networks, no additional levels are typicallyneeded because triple, or higher order points in the projection are notnecessary to allow the channels within the structure to cross over orunder each other and thus cross each other in space without physicalintersection of the “crossing” channels within the structure.

[0056]FIG. 1a illustrates one exemplary embodiment of an essentiallyinfinite number of microfluidic network structures that can be producedaccording to the invention. Microfluidic network structure 100 includesa series of interconnected channels providing seven non-fluidicallyinterconnected fluid flow paths. The channels are arranged in a “basketweave” arrangement. Channel system 100, as illustrated, includes threenon-fluidically interconnected fluid flow paths, 102, 104, and 106arrayed within planes parallel to the y-z coordinate plane, and fournon-fluidically interconnected flow paths 108, 110, 112, and 114 arrayedwithin planes parallel to the x-z coordinate plane. Each fluid flow pathof the structure comprises a series of interconnected channels (e.g.fluid flow path 102 comprises interconnected channels 113, 124, 126,116, 118, 120, 128, 122 and 123 within structure 100).

[0057] Flow path 102, for example, includes two channels 116 and 122disposed within the first, lower level of structure 100 and two channels120 and 124 disposed within the second, upper level of the structure.Flow path 102 also includes a number of connecting channels, e.g. 118,126, and 128 traversing a third, intermediate level of the structure andinterconnecting channels contained in the first, lower level and second,upper level of the structure. The microfluidic network provided bystructure 100 is truly three-dimensional because it cannot be producedby a two-dimensional structure comprising a series of interconnectedchannels disposed within a single plane or any stack or array of suchstructures. In other words, network 100 includes channels disposedwithin the first, lower level of the structure that are non-parallel tochannels disposed within the second, upper level of the structure (e.g.channel 116 of fluid flow path 102 and channel 130 of fluid flow path110). Another way to describe the three-dimensionality of network 100,and distinguish the network from those realizable in two-dimensionalsystem, is to point out, that, for example, flow path 102 comprises aseries of non-colinear interconnected channels disposed within a firstplane of the structure, which is parallel to the y-z coordinate plane,and a second fluid flow path, for example, fluid flow path 108, isdisposed within a second plane (parallel to the x-z coordinate plane asshown) that is not parallel with the first plane. Yet another way inwhich the microfluidic networks provided according to the inventiondiffer from those realizable with two-dimensional systems is that theinventive microfluidic systems can include a fluid flow path thereinhaving a longitudinal axis, defining a direction of bulk fluid flowwithin the flow path, that is not disposed within any single plane inspace, nor is disposed within any a surface that is parallel to anysurface (such as surface 132 or 134) of the microfluidic structure.

[0058] A “level” of a structure, as used herein, refers to a plane orcurved surface within the structure, typically parallel to a top surfaceand a bottom surface of the structure, which can have a channel orseries of channels disposed therein and/or penetrating therethrough. Itshould be understood that in the discussion and figures illustratedbelow, the microfluidic network structures are generally shown as havingplanar surfaces (e.g. surfaces 132 and 134), such that the levels withinthe structure are planar; however, many of the structures, as describedin more detail below, are fabricated from flexible and/or elastomericmaterials that are capable of being bent, twisted, or distorted from theillustrated planar configurations. For such embodiments, the “levels”within the structure will comprise curved surfaces that are parallel tothe distorted planar surfaces of the structure, and any discussionherein with regard to “levels” of the structures should be understood toencompass such curved surfaces as well as the planar surfacesillustrated. “Parallel,” when used in the context of comparing thetopology of two surfaces in space, has its common mathematical meaningreferring to the two surfaces being everywhere spaced apart from eachother equidistantly.

[0059] “Non-fluidically interconnected” fluid flow paths, as usedherein, refers to fluid flow paths each comprising one channel ormultiple, fluidically interconnected channels, where the channels ofdifferent flow paths do not intersect and are physically isolated fromeach other within the structure so that they can not communicate fluidbetween each other through bulk mixing of fluid streams. A “fluid flowpath” as used herein refers to one channel or a series of two or moreinterconnected channels providing a space within the microfluidicstructure able to contain fluid or through which fluid can continuouslyflow. Each fluid flow path of the structure includes at least oneopening thereto able to be placed in fluid communication with theenvironment external to the microfluidic structure and some preferredembodiments of fluid flow paths include at least two openings able to beplaced in fluid communication with the environment external to themicrofluidic structure, thus providing an inlet and an outlet. A“channel” as used herein refers to a flow path or continuous segment ofa flow path, which is disposed within one or more levels of themicrofluidic network structure and/or penetrates through one or morelevels of the microfluidic network structure. “Interconnected channels,”as used herein, refers to two or more channels within the structure thatare able to communicate fluid between and through each other. A“non-linear” flow path and/or channel, as used herein, refers to suchflow path or channel having a longitudinal axis that deviates from astraight line along its length by more than an amount equal to theminimum cross-sectional dimension of the channel or flow path. A“longitudinal axis” of a channel or flow path as used herein refers toan axis disposed along the entire length of such channel or flow path,which is coextensive with and defined by the geometric centerline of thedirection of any bulk fluid which would flow through the channel or flowpath should such channel or flow path be configured for fluid flowtherethrough. For example, a linear or “straight” channel would tend tohave a longitudinal axis that is essentially linear, while a fluid flowpath comprising a series of such straight channels that are fluidicallyinterconnected can have a longitudinal axis, comprising theinterconnected longitudinal axes of the individual interconnectedchannels forming the fluid flow path, which is “non-linear.” A channelwhich is “disposed within,” “disposed in,” “contained within,” or“contained in” a level or multiple levels of the structure refers hereinto such channel having a longitudinal axis that is coplanar with or, inthe case of a level defined by a curved surface, is lying along acontour of the surface, of the level(s) in which it is disposed orcontained. A channel that “penetrates,” “penetrates through,” or“traverses” a level or multiple levels of the structure refers herein tosuch channel having a longitudinal axis that is non-coplanar with or, inthe case of a level defined by a curved surface, is not lying along acontour of the surface of the level(s) such that the longitudinal axisof such channel is non-parallel with any line that can be disposedwithin the level.

[0060] Fluid flow path 102 of microfluidic network 100 communicates withthe external environment through an inlet opening 136 in fluidcommunication with bottom surface 134 and an outlet opening 138 in fluidcommunication with upper surface 132. The other fluid flow paths of thenetwork have similar inlet and outlet openings, as illustrated.

[0061] The channels of the microfluidic networks provided according tothe invention have at least one cross-sectional dimension that does notexceed about 500 μm, in other embodiments does not exceed about 250 μm,in yet other embodiments does not exceed about 100 μm, in otherembodiments does not exceed about 50 μm, and in yet other embodimentsdoes not exceed about 20 μm. A “cross-sectional dimension,” when used inthe above context, refers to the smallest cross-sectional dimension fora cross-section of a channel taken perpendicular to the longitudinalaxis of the channel. While the channels of network 100 havecross-sectional dimensions that are essentially equal to each other, inother embodiments, the channels can have unequal cross-sectionaldimensions, and some channels can have depths within the structuresufficiently great so that they are disposed in two or all three levelsof the structure, instead of being disposed in only a single level, asillustrated. In addition, while in network 100 the channels are straightand linear, in other embodiments the channels can be curved within thelevel(s) in which they are disposed.

[0062] The double points formed where the channels of the fluid flowpaths of network 100 cross over each other are more clearly seen in thetwo-dimensional perpendicular projection shown in FIG. 1b. FIG. 1b showsmicrofluidic network 100 as projected onto the y-x plane as viewed inthe negative z-axis direction. Crossover double point 140, for example,represents the double point defining the cross over of channel 130 offluid flow path 110 and channel 116 of fluid flow path 102. In general,microfluidic networks provided according to the invention having fluidflow paths including channels that “cross over” each other refers tostructures including channel networks wherein a perpendicular projectionof the channels onto a surface defining a level of the structure, inwhich either of the channels are disposed, at least partially overlapeach other. A “perpendicular projection” refers to a projection in adirection that is perpendicular or normal to the surface being projectedupon. “At least partially overlap” or “at least partially overlapping,”as used herein when referring to projections of channels which crossover each other, refers to the two-dimensional projection of thechannels intersecting each other, as shown by point 140 in FIG. 1b, or,if, for example, the channels are arranged in a parallel direction withrespect to each other within the network structure, to their being atleast partially superimposed upon each other in the two-dimensionalprojection.

[0063] While the three-dimensional microfluidic network structuresdescribed herein could potentially be fabricated via conventionalphotolithography, microassembly, or micromachining methods, for example,stereolithography methods, laser chemical three-dimensional writingmethods, or modular assembly methods, as described in more detail below,the invention also provides improved fabrication methods for producingthe inventive structures involving replica molding techniques forproducing individual layers which comprise one or more of the levels ofthe structures, as discussed above. As described in more detail below,such layers are preferably molded utilizing mold masters having variousfeatures on their surface(s) for producing channels of the structure. Insome preferred embodiments, the features are formed via aphotolithography method, or can themselves comprise a molded replica ofsuch a surface.

[0064] The microfluidic network structures produced by the inventivemethods described herein can potentially be formed from any materialcomprising a solid material that comprises a solidified form of ahardenable liquid, and, in some embodiments, the structures can beinjection molded or cast molded. As will be described in more detailbelow, preferred hardenable liquids comprise polymers or precursors ofpolymers, which harden upon, or can be induced to harden during, moldingto produce polymeric structures. For reasons described in more detailbelow, particularly preferred polymeric materials for forming themicrofluidic networks according to the invention comprise elastomericmaterials.

[0065] For structures produced according to the preferred methodsdescribed herein, the microfluidic networks provided according to theinvention will typically be comprised of at least one discrete layer ofpolymeric material, and other embodiments will be comprised of at leasttwo discrete layers of polymeric material, and in yet other embodimentswill be comprised of three or more discrete layers of polymericmaterial. A “discrete layer” of material as used herein refers to aseparately formed subcomponent structure of the overall microfluidicstructure, which layer can comprise and/or contain one, two, or three,or more levels of the overall channel network of the microfluidicstructure. As described and illustrated in more detail below, thediscrete layers of the structure can be stacked together to form athree-dimensional network, or multiple three-dimensional networks, ifdesired, and can also be, in some embodiments, placed between one ormore support layers or substrate layers in order to enclose andfluidically seal channels of the lower and upper levels of themicrofluidic structure.

[0066] As described in more detail below, the methods for producingmicrofluidic network structures provided by the invention can, in someembodiments, produce discrete layers comprising a single level of theoverall structure, wherein the three-dimensional network structure isformed by forming a first layer including a series of channels disposedtherein, forming a second layer including a second series of channelsdisposed therein, and forming a third layer having connecting channelstraversing the layer, and subsequently stacking the third layer betweenabove-mentioned first and second layers and aligning the layers withrespect to each other to achieve the overall desired three-dimensionalnetwork structure. In another embodiment, the microfluidic networkstructure includes two channel-containing layers: a first discrete layercontaining both a first level, including a series of channels disposedtherein, and a third, intermediate level of the structure including theconnecting channels traversing the level; and a second discrete layerincluding the second level of the structure, having a second series ofchannels disposed therein. In such a method the first discrete layer andthe second discrete layer are stacked and aligned with respect to eachother to produce the overall desired three-dimensional microfluidicnetwork structure. And in yet a third embodiment, all three levels ofthe microfluidic network structure can be produced in a single discretelayer, the layer comprising a three-level microfluidic membranestructure.

[0067]FIGS. 2a and 2 b illustrate a microfluidic structure 150 having analternative three-dimensional arrangement of channels therein.Microfluidic network 150 includes two non-fluidically interconnectedflow paths 152 and 154. Fluid flow path 152 comprises a series ofinterconnected channels 156, 158, 160, 162 and 164, which are non-linearand which define a plane parallel to the y-z coordinate plane. Channels156 and 164 are disposed within a first, lower level of the structure,and channel 160 is disposed within a second, upper level of thestructure. Connecting channel 158 traverses a third, intermediate levelof the structure from the first, lower level to the second, upper leveland fluidically interconnects channel 156 to channel 160. Similarly,connecting channel 162 traverses the third, intermediate level of thestructure connecting channel 164 and channel 160. Flow path 152 isconnected in fluid communication with the external environment via inletopening 168 in side wall 170 an outlet opening 172 in side wall 174.Fluid flow path 154 comprises a single channel 176 disposed within thefirst, lower level of the structure, and is interconnected to theenvironment via inlet opening 178 in side wall 180 an outlet opening 182in side wall 190. The perpendicular projection of the microfluidicchannel network, onto the first, lower level of the structure isillustrated in FIG. 2b. FIG. 2b shows double point 192 where channel 160of fluid flow path 152 crosses over channel 176 of fluid flow path 154.

[0068]FIGS. 3a and 3 b illustrate yet another simple microfluidicnetwork provided according to the invention but not achievable with aconventional two-dimensional microfluidic network structure.Microfluidic network 200 includes a single fluid flow path 202. Fluidflow path 202 is comprised of a first channel 204 disposed within afirst, lower level of the structure; a second channel 206 disposedwithin a second, upper level of the structure; and a connecting channel208 traversing a third, intermediate level of the structure andfluidically interconnecting channels 204 and 206. Channel 204 disposedwithin the first level of the structure and channel 206 disposed withinthe second level of the structure are non parallel to each other and, inthe illustrated embodiment, happen to be perpendicular to each other.FIG. 3b illustrates the perpendicular projection of microfluidic network200 onto the first, lower level structure along the negative z-axisdirection. As illustrated, microfluidic network 200 does not include anycrossover points in the projection.

[0069] As previously discussed, a microfluidic network need only includethree levels therein (a first and a second level including channelsdisposed therein such that their longitudinal axes are coplanar with asurface defining the level and a third intermediate level having one ormore connecting channels passing therethrough fluidically connecting thechannels of the first level and the second level) in order to provideany arbitrarily complex network of channels that pass over and under oneanother. However, certain potentially desirable geometric configurationsof channels may require more than the three levels contained within thestructures discussed and illustrated above. For example, if it isdesired to produce a microfluidic network having channels disposedwithin three or more non-coplanar levels of the structure, additionallevels are needed. In general, the number of levels required formicrofluidic structures produced according to the invention required toproduce n levels, each level having channels disposed therein such thattheir longitudinal axis are coplanar with the level, requires a total of2n−1 total levels in the structure. Thus, for the previously illustratedembodiments having two levels therein in which channels are disposed,each structure requires a total of three levels to form the overallnetwork structure (an upper and lower level in which the channels aredisposed and an intermediate level through which the connecting channelspass).

[0070]FIGS. 4a and 4 b illustrate one embodiment of a microfluidicstructure, producible according to the methods of the inventiondescribed below, including therein three levels having channels disposedtherein such that their longitudinal axes are coplanar with each of thelevels, and a total of five levels overall. Structure 220 includes amicrofluidic network comprising a fluid flow path 222 arranged as a coilsurrounding a second fluid flow path 224. Such an arrangement may beespecially useful for particular microfluidic applications involving,for example, heat transfer or mass transfer between components containedwithin fluid flow paths 222 and 224, or for embodiments whereelectrical, magnetic, optical or other environmental interaction betweenmaterials in the respective flow paths is desired.

[0071] The first, lower level of structure 220 includes disposed thereinchannels 226, 228, 230, and 232 of coil flow path 222. The second levelfrom the bottom of structure 220 includes disposed therethrough thelowermost region 234 of connecting channels 236, 238, 240, 242, 244,246, and 248 of fluid flow path 222. The third level from the bottom ofstructure 220 includes channel 250 of fluid flow path 224 disposedtherein and also includes intermediate region 251 of the connectingchannels. The fourth level from the bottom of structure 220 includes,traversing therethrough, upper regions 252 of the connecting channels,and the uppermost level of structure 220 includes disposed thereinchannels 254, 256, 258 and 260 of flow path 222.

[0072]FIG. 4b illustrates the perpendicular projection of microfluidicnetwork 220 onto a surface coplanar with the first, lowermost level ofthe structure that is parallel to the y-x coordinate plane, as viewed inthe negative z direction. As illustrated, structure 220 includes 8double point crossovers 264, 266, 267, 268, 269, 270, 272, and 274 whereeither flow path 224 crosses over a channel of flow path 222 (e.g.crossover points 264, 267, 269, and 272), or where channel 250 of flowpath 224 crosses under a channel of fluid flow path 222, (for example,crossover point 266, 268, 270, and 274.) It should be evident that thefive level structure illustrated by structure 220, in alternativeembodiments, can have flow paths therein comprising a series ofinterconnected channels arranged so as to yield higher order crossoverpoints than the double points illustrated. For example, in otherembodiments, a five level structure can have channels disposed thereinincluding triple point crossovers wherein a perpendicular projectiononto a surface coplanar with a level of the structure includes pointswhere three levels of channels intersect (i.e., where a channel disposedin the lowermost level, a channel disposed in the third, intermediatelevel, and a channel disposed in the uppermost level overlap and/orintersect each other in the two-dimensional projection).

[0073] As discussed above, the present invention also provides a varietyof methods providing relatively simple and low cost fabricationtechniques for producing the inventive microfluidic structures describedherein. The preferred methods provided according to the invention anddescribed below are based upon utilizing a hardenable liquid to createreplica molded structures that comprise, or are assembled with otherreplica molded structures to form, the three-dimensional microfluidicnetwork structures provided by the invention.

[0074]FIGS. 5a-5 c illustrate a first embodiment of a method for formingthe inventive microfluidic structures by utilizing a replica moldingprocess provided by the invention. The method illustrated by FIGS. 5a-5c involves forming a number of replica molded layers from a hardenableliquid, each of which structures comprises a single level of the overallmicrofluidic network. Following the fabrication of each of the replicamolded structures comprising layers of the overall microfluidic networkstructure, the layers are stacked upon each other, aligned with respectto each other so that the respective molded features in the layerscreate the desired and predetermined microfluidic network pattern, and,optionally, the layers can be permanently sealed to each other and/or toone or more substrate layers, which substrate layers do not comprise alevel of the overall microfluidic structure, in order to yield afinished microfluidic network structure having a desired configuration.

[0075] Step 1 as illustrated in FIG. 5a involves forming a first layerof the structure comprising, for example, a first, lower level of themicrofluidic network. Of course, in other embodiments, layers comprisingan upper or intermediate level of the structure can be molded before orat the same time a lower layer is molded. In general, the order of themolding steps is not particularly critical and the various layers of theoverall structure can be molded in any order that is desired orconvenient. In the illustrated embodiment, a lower mold master 300 isprovided having a series of topological features 302 protruding from anupper surface 304 of the lower mold master. A second mold master 306having a flat, featureless surface 308 facing surface 304 of mold master300 is provided and placed in contact with an upper surface oftopological features 302 of mold master 304. Disposed between moldmasters 304 and 306 is a layer of hardenable liquid 310, which uponsolidification forms a replica molded layer including therein aplurality of channels, formed by topological features 302 of mold master304, which, channels, in preferred embodiments, pass completely throughthe thickness of the entire layer of liquid 310, upon hardening, thusforming a membrane structure comprised of the hardened liquid.

[0076] Mold master 300, having positive, high-relief topologicalfeatures 302 formed on a surface 304 thereof comprises, in somepreferred embodiments, a substrate that has been modified, for example,via photolithography or any suitable micromachining method apparent tothose of ordinary skill in the art. Topological features 302 are shaped,sized, and positioned to correspond to a desired arrangement of channelsin the level of the overall microfluidic network structure being formedby the mold master. In one preferred embodiment, mold master 300comprises a silicon wafer having a surface 304 that has been viaphotolithography utilizing a photomask having a pattern therewithincorresponding to a desired pattern of topological features 302.Techniques for forming positive relief patterns of topological featureson silicon, or other materials, utilizing photolithography andphotomasks, are well known and understood by those of ordinary skill inthe art and, for example, are described in Qin, D., et al. “RapidPrototyping of Complex Structures with Feature Sizes Larger Than 20microns,” Advanced Materials, 8(11):pp.917-919 and Madou, M.,Fundamentals of Microfabrication, CRC Press, Boca Raton, Fla., (1997),both incorporated herein by reference.

[0077] In a particularly preferred embodiment, mold master 300 comprisesa silicon or other substrate, which has been spincoated with one or morelayers of a commercially available polymeric photoresist material. Insuch preferred embodiments, topological features 302 can be easily,conveniently, and accurately formed in the layer(s) of photoresistforming surface 304 of substrate 300 via exposure of photoresist toradiation through a photomask and subsequent development of thephotoresist material to remove photoresist material from the surface andregions surrounding features 302 thus leaving behind topologicalfeatures 302 in positive relief. A variety of positive and negativephotoresists can be utilized for such purposes and are well known tothose of ordinary skill in the art.

[0078] One particularly preferred method for forming topologicalfeatures 302 on a surface of a substrate coated with one or more layersof photoresist is described in more detail below in the context of FIG.8. The photomask utilized, as described above, provides a patterntherein able to selectively block radiation reaching the layer(s) ofphotoresist so that, upon development of the layer, a pattern oftopological features will be formed, which features correspond to adesired arrangement of channels within the replica molded layer. Suchpatterns can be designed with the aid of any one of a number ofcommercially available computer aided design (CAD) programs, as would beapparent to those of ordinary skill in the art.

[0079] Mold master 306 can be comprised of the same material as moldmaster 300; however, in preferred embodiments, mold master 306 is formedof an elastomeric material, for example, an elastomeric polymer. Moldmaster 306 is, in preferred embodiments, formed of an elastomericmaterial because the elastomeric nature of the mold master enables animproved seal at the interface of surface 308 of mold master 306 and theupper surfaces of topological features 302 of lower mold master 300 tobe formed so as to essentially completely exclude hardenable liquid 310from the interface between the topological features 302 and surface 308of mold master 306. This preferred (“sandwich”) method enables, upon thehardening of hardenable liquid 310, the production of a membranecomprised of the hardened fluid having channels disposed therein whichcompletely traverse the entire thickness of the membrane and which arenot blocked by a thin layer of hardened liquid.

[0080] For some embodiments, it is also desirable that upper mold master306 be transparent in order to be able to visualize topological features302 during the molding process. Alternatively, in other embodiments,upper mold master 306 can comprise a rigid, non-elastomeric material andlower mold master 300, including topological features 302 forming thechannels of the molded structure, can be formed of an elastomericmaterial. In such an embodiment, the elastomeric mold master havingpositive relief topological features disposed on its surface ispreferably itself formed as a molded replica of a pre-master having asurface including a plurality of negative, low-relief features therein,which form the positive relief features in the elastomeric mold masterupon creating a replica mold of the pre-master surface. In yet otherembodiments, the upper and lower mold masters of the invention can bothcomprise elastomeric materials and can be formed of the same, ordifferent elastomeric materials. In addition, although less preferred,upper mold master 306 can be eliminated entirely and hardenable fluid310 may simply be spuncast onto surface 304 of lower mold master 300 toa thickness corresponding to the height of topological features 302.Such method is generally less preferred for producing molded membranesaccording to the invention because it is generally desired that theuppermost and lowermost surfaces of the membrane be as flat and smoothas possible to enable conformal sealing and prevention of leakage uponassembly of the layers into the overall microfluidic network structure.

[0081] In preferred embodiments, hardenable liquid 310 is placed uponsurface 304 of lower mold master 300 in an amount sufficient to form alayer over the region of surface 304 including topological features 302,corresponding to the channel structure in the layer to be formed, whichlayer having a thickness at least equal to the height of topologicalfeatures 302 above surface 304. Subsequent to placing liquid 310 onsurface 304, the method involves bringing surface 308 of upper moldmaster 306 into contact with the upper surface of features 302. Inalternative embodiments, a lower mold master and upper mold master canbe brought into contact prior to addition of the hardenable liquid, andthe hardenable liquid can be applied to the region between the facingsurfaces of the mold masters by adding a sufficient amount in the regionof the space between the upper mold master and lower mold master aroundtheir periphery (e.g. periphery 312), and subsequently allowinghardenable liquid 310 to flow into the space surrounding the topologicalfeatures of the mold master(s) via capillary action. Such method forutilizing capillary action for creating a molded replica structure asdescribed in detail in commonly owned, copending U.S. patent applicationSer. No. 09/004,583 entitled “Method of Forming Articles IncludingWaveguides Via Capillary Micromolding and Microtransfer Molding,” andInternational Pat. Publication No. WO 97/33737, each incorporated hereinby reference.

[0082] Hardenable liquid 310 can comprise essentially any liquid knownto those of ordinary skill in the art that can be induced to solidify orspontaneously solidifies into a solid capable of containing andtransporting fluids contemplated for use in and with the microfluidicnetwork structures. In preferred embodiments, hardenable liquid 310comprises a polymeric liquid or a liquid polymeric precursor (i.e. a“prepolymer”). Suitable polymeric liquids can include, for example,thermoplastic polymers, thermoset polymers, or mixture of such polymersheated above their melting point; or a solution of one or more polymersin a suitable solvent, which solution forms a solid polymeric materialupon removal of the solvent, for example, by evaporation. Such polymericmaterials, which can be solidified from, for example, a melt state or bysolvent evaporation, are well known to those of ordinary skill in theart.

[0083] In preferred embodiments, hardenable liquid 310 comprises aliquid polymeric precursor. Where hardenable liquid 310 comprises aprepolymeric precursor, it can be, for example, thermally polymerized toform a solid polymeric structure via application of heat to mold master300 and/or mold master 306; or, in other embodiments, can bephotopolymerized if either mold master 300 or mold master 306 istransparent to radiation of the appropriate frequency. Curing andsolidification via free-radical polymerization can be carried out aswell. These and other forms of polymerization are known to those ofordinary skill in the art and can be applied to the techniques of thepresent invention without undue experimentation. All types ofpolymerization, including cationic, anionic, copolymerization, chaincopolymerization, cross-linking, and the like can be employed, andessentially any type of polymer or copolymer formable from a liquidprecursor can comprise hardenable liquid 310 in accordance with theinvention. An exemplary, non-limiting list of polymers that arepotentially suitable include polyurethane, polyamides, polycarbonates,polyacetylenes and polydiacetylenes, polyphosphazenes, polysiloxanes,polyolefins, polyesters, polyethers, poly(ether ketones), poly(alkalineoxides), poly(ethylene terephthalate), poly(methyl methacrylate),polystyrene, and derivatives and block, random, radial, linear, orteleblock copolymers, cross-linkable materials such as proteinaceousmaterials and/or blends of the above. Gels are suitable wheredimensionally stable enough to maintain structural integrity uponremoval from the mold masters, as described below. Also suitable arepolymers formed from monomeric alkylacrylates, alkylmethacrylates,alpha-methylstyrene, vinyl chloride and other halogen-containingmonomers, maleic anhydride, acrylic acid, acrylonitrile, and the like.Monomers can be used alone, or mixtures of different monomers can beused to form homopolymers and copolymers. The particular polymer,copolymer, blend, or gel can be selected by those of ordinary skill inthe art using readily available information and routine testing andexperimentation so as to tailor a particular material for any of a widevariety of potential applications.

[0084] According to some preferred embodiments of the invention,hardenable liquid 310 comprises a fluid prepolymeric precursor whichforms an elastomeric polymer upon curing and solidification. A varietyof elastomeric polymeric materials are suitable for such fabrications,and are also suitable for forming mold masters, for embodiments whereone or both of the mold masters is composed of an elastomeric material.A non-limiting list of examples of such polymers includes polymers ofthe general classes of silicone polymers, epoxy polymers, and acrylatepolymers. Epoxy polymers are characterized by the presence of athree-membered cyclic ether group commonly referred to as an epoxygroup, 1, 2-epoxide, or oxirane. For example, diglycidyl ethers ofbisphenol A can be used, in addition to compounds based on aromaticamine, triazine, and cycloaliphatic backbones. Another example includesthe well-known Novolac polymers. Examples of silicone elastomerssuitable for use according to the invention include those formed fromprecursors including the chlorosilanes such as methylchlorosilanes,ethylchlorosilanes, and phenylchlorosilanes, and the like. Aparticularly preferred silicone elastomer is polydimethylsiloxane(PDMS). Exemplary polydimethylsiloxane polymers include those sold underthe trademark Sylgard by Dow Chemical Co., Midland, Mich., andparticularly Sylgard 182, Sylgard 184, and Sylgard 186.

[0085] Silicone polymers, for example, PDMS, are especially preferredfor use in the invention because they have several desirable beneficialproperties simplifying fabrication of the microfluidic networkstructures, described herein. First, such materials are inexpensive,readily available, and can be solidified from a prepolymeric liquid viacuring with heat. For example, PDMSs are typically curable by exposureof the prepolymeric liquid to temperatures of about, for example, 65° C.to about 75° C. for exposure times of about, for example, 1 hour.Second, silicone polymers, such as PDMS, are elastomeric and are thususeful for forming certain of the mold masters used in some embodimentsof the invention. In addition, microfluidic networks formed fromelastomeric materials can have the advantage of providing structureswhich are flexible and conformable to the shape of a variety ofsubstrates to which they may be applied, and elastomeric networks canprovide reduced resistance to fluid flow for a given applied pressuredrop, as compared to non-elastomenrc structures, and can also be moreeasily fabricated to include active elements therein, for exampleintegrated valves and pumping elements, which elements can utilize theflexibility and elasticity of the material for their performance.

[0086] Another distinct advantage for forming the inventive microfluidicnetworks from silicone polymers, such as PDMS, is the ability of suchpolymers to be oxidized, for example by exposure to an oxygen-containingplasma such as an air plasma, so that the oxidized structures contain attheir surface chemical groups capable of cross-linking to other oxidizedsilicone polymer surfaces or to the oxidized surfaces of a variety ofother polymeric and non-polymeric materials. Thus, membranes, layers,and other structures produced according to the invention utilizingsilicone polymers, such as PDMS, can be oxidized and essentiallyirreversibly sealed to other silicone polymer surfaces, or to thesurfaces of other substrates reactive with the oxidized silicone polymersurfaces, without the need for separate adhesives or other sealingmeans. In addition, microfluidic structures formed from oxidizedsilicone polymers can include channels having surfaces formed ofoxidized silicone polymer, which surfaces can be much more hydrophilicthan the surfaces of typical elastomeric polymers. Such hydrophilicchannel surfaces can thus be more easily filled and wetted with aqueoussolutions than can structures comprised of typical, unoxidizedelastomeric polymers or other hydrophobic materials.

[0087] In addition to being irreversibly sealable to itself, oxidizedPDMS can also be sealed irreversibly to a range of oxidized materialsother than itself including, for example, glass, silicon, silicon oxide,quartz, silicon nitride, polyethylene, polystyrene, glassy carbon, andepoxy polymers, which have been oxidized in a similar fashion to thePDMS surface (for example, via exposure to an oxygen-containing plasma).Oxidation and sealing methods useful in the context of the presentinvention are described in more detail below and also in Duffy et al.,Rapid Prototyping of Microfluidic Systems and Polydimethylsiloxane,Analytical Chemistry, Vol. 70, pages 474-480, 1998, incorporated hereinby reference.

[0088] For clarity and simplicity, the discussion below involving theinventive methods for forming microfluidic structures according to theinvention in many instances makes specific reference to a preferredembodiment wherein the layers comprising the structure and/or one ormore mold masters are formed from a hardenable liquid comprising a fluidprepolymer of PDMS. It should be understood, as the discussion abovemakes clear, that such reference is pure exemplary, and a wide varietyof other materials can be utilized in place of or in addition to PDMS toachieve the various objects, features, and benefits of the presentinvention, as would be apparent to those of ordinary skill in the art.

[0089] Referring again to FIG. 5a, in Step 2, PDMS, comprisinghardenable liquid 310, is cured and solidified, for example byapplication of heat to raise the temperature of the PDMS prepolymer tobetween about 65° C. to about 75° C. for about 1 hour, as describedabove. In order to prevent seepage of the PDMS between surface 308 andthe upper surface of topological features 302, it is preferred to applypressure to one or both of lower surface 314 of mold master 300 andupper surface 316 of mold master 306. It has been found, within thecontext of the invention, that a pressure of approximately between about10-100 g/mm² (100-1,000 kPa) or greater is generally sufficient toprevent PDMS prepolymer from seeping between topological features 302and surface 308 so as to cause blockage of subsequent channels formedwithin the cured membrane.

[0090] Step 3 involves peeling the cured membrane from one or both ofmold master 300 and 306. In preferred embodiments, as discussed above,materials are selected for mold master 300, mold master 306, andhardenable liquid 310, which allow removal of the solidified membraneupon solidification of the hardenable liquid without destruction of themolded structure. In especially preferred embodiments, because asolidified layer is typically thin and fragile (for example, layer 318can vary in thickness from about 20 μm to about 1 mm), mold master 300and mold master 306 are selected or treated such that layer 318 adheresto the surface of one of the mold masters more strongly than to thesurface of the other mold master. Such differential adhesion allows themold masters to be peeled apart such that the fragile molded layer 318remains adherent to and is supported by one or the other of the moldmasters. Such differential adhesion of layer 318 can be created byselecting materials comprising mold master 306 and surface 304 of moldmaster 300 having different chemical properties such that thenon-covalent interfacial adhesion between layer 318 and surface 304differs from that between layer 318 and surface 308. Those of ordinaryskill in the art can readily determine appropriate materials forcomprising hardenable liquid 310, mold master 300, and mold mater 306and/or surface treatments which can be applied to either or both of themold masters that allow for differences in non-covalent interfacialadhesion between layer 318 and the surfaces of the mold masters,enabling layer 318 to be selectively removed from one of the surfaceswhile remaining adherent to the other. Interfacial free energies for awide variety of materials are readily available to those of ordinaryskill in the art and can be utilized, along with routine screeningtests, for example measuring forces required to peel apart variouscombinations of materials, by those of ordinary skill in the art toreadily select a combination of materials, without undueexperimentation, for enabling layer 318 to be selectively removed fromthe surface of one mold master while remaining adherent to and supportedby the surface of the other mold master.

[0091] For example, in the illustrated embodiment, lower mold master 300includes an upper surface 304 comprising a negative photopolymer(SU-8-50, Microlithography Chemical Corp., Newton, Mass.), upper moldmaster 306 comprises oxidized PDMS, and hardenable fluid 310 comprises aPDMS prepolymer. Also in the illustrated embodiment, surfaces 308 and304, before contact with fluid 310 were silanized to facilitate theremoval of PDMS replica layer 318 after curing. In an exemplaryembodiment, the masters were silanized by exposing the surfaces to achlorosilane vapor, for example a vapor containingtridecafluoro-1,1,2,2-tetrahydrooctal-1-trichlorosilane. PDMS replicalayer 318 adheres more strongly to silanized PDMS mold master 306 thanto silanized surface 304 of mold master 300 and remains supported by andattached to mold master 306 upon applying a peeling force tending toseparate the two mold masters, resulting in molded replica layer 318remaining adherent and supported by mold master 306, as illustrated inStep 3. In an alternative embodiment, instead of utilizing a silanizedPDMS layer for mold master 306 in combination with silanized mold master300, as described above, mold master 306 can comprise a layer or sheetof a material having a very low interfacial free energy, for exampleTeflon™ (polytetrafluoroethylene (PTFE)). In such an embodiment, replicamolded layer 318 will tend to remain adherent to mold master 300 uponapplying a peeling force tending to separate mold master 306 and moldmaster 300.

[0092] Step 4 of FIG. 5a illustrates an optional step comprisingconformally contacting molded replica layer 318, supported by moldmaster 306, with a lower substrate layer 320, and, optionally,irreversibly sealing lower surface 319 of layer 318 to the upper surface322 of substrate 320. In the illustrated embodiment, substrate 320comprises a PDMS slab having a flat upper surface 322. Both lowersurface 319 of layer 318 and upper surface 322 of substrate 320 havebeen oxidized, for example by exposure to an air plasma in a plasmacleaner, as discussed above and in more detail below, prior to bringingthe surfaces into contact, so that when brought into conformal contact,an irreversible seal spontaneously forms between surface 319 and surface322 providing a fluid-tight seal at the bottom of channels 321 in layer318. Exposure of the PDMS surfaces to the oxygen-containing plasma isbelieved to cause the formation of Si—OH groups at the surface of thePDMS, which react with other Si—OH groups to form bridging, covalentsiloxane (Si—O—Si) bonds by a condensation reaction between the twooxidized PDMS surfaces.

[0093] In alternative embodiments, where it is not desired topermanently seal layer 318 to substrate 320, the surfaces may not beoxidized so that they do not irreversibly seal to each other but rathermay simply be brought into conformal contact with each other, whichconformal contact between the two essentially flat planar surfaces canbe sufficient, for microfluidic applications involving vacuum or lowpressures, to form a fluid-tight seal. Also, in some applications, suchas microcontact surface patterning with the inventive microfluidicnetworks as described in more detail below, it may be desirable toprovide a “patterning” surface of the microfluidic network havingchannels therein which are not sealed by a substrate, and which can bebrought into contact with a material surface in order to form on thesurface a pattern defined by the channels in the “patterning” surface ofthe microfluidic network.

[0094] In yet other embodiments, substrate 320 can comprise a materialdifferent from one or both of molded layer 318 and mold master 306, forexample, a material other than PDMS. In some such embodiments, substrate320 can comprise, for example, the surface of a silicon wafer ormicrochip, or other substrate advantageous for use in certainapplications of the microfluidic network provided according to theinvention. Molded layer 318 can, as described above, be irreversiblysealed to such alternative substrates or may simply be placed inconformal contact without irreversible sealing. For embodiments where itis desired to irreversibly seal a molded replica layer 318 comprisingPDMS to a substrate 320 not comprising PDMS, it is preferred thatsubstrate 320 be selected from the group of materials other than PDMS towhich oxidized PDMS is able to irreversibly seal (e.g., glass, silicon,silicon oxide, quartz, silicon nitride, polyethylene, polystyrene, epoxypolymers, and glassy carbon surfaces which have been oxidized). Forembodiments involving hardenable liquids other than PDMS prepolymers,which form molded replica layers not able to be sealed via the oxidationmethods described above, when it is desired to irreversibly seal suchlayers to each other or to a substrate, alternative sealing means can beutilized, as would be apparent to those of ordinary skill in the art,including, but not limited to, the use of separate adhesives, thermalbonding, solvent bonding, ultrasonic welding, etc.

[0095] Step 5 illustrated in FIG. 5a comprises the removal of upper moldmaster 306 to expose flat, top surface 317 of molded replica 318 thusyielding a first, lower level of the overall microfluidic networkstructure having a series of channels 321 disposed in a desired patterntherein. In an alternative embodiment to the illustrated membranesandwich method for forming membrane layer 318, in Step 1 a moldedreplica can be formed by placing mold master 300 in the bottom of a dishor other container having a depth in excess of the height of topologicalfeatures 302 and filling the container to a level in excess of theheight of features 302 with a hardenable liquid, such as PDMSprepolymer. Upon curing and removing the cured structured from thecontainer and from mold master 300, a structure similar to that obtainedat the end of Step 5 is formed, except comprising channels that do notpenetrate through the entire thickness of the molded replica layer. Suchan embodiment is described in further detail in the context of thefabrication method illustrated in FIG. 7 below. In addition, asillustrated in FIG. 5, to facilitate the stacking and alignment ofadditional molded replica layers comprising the second, third, and anyhigher levels of the microfluidic structure, lower layer 318 can betrimmed such that it is essentially uniform in thickness and has adesired overall size and perimeter shape.

[0096]FIG. 5b illustrates the formation of a second molded replica layercomprising the third, intermediate level of a microfluidic networkstructure containing therein connecting channels as previouslydescribed. Steps 6-8 are essentially similar to Steps 1-3 describedabove in the context of FIG. 5a, except that lower mold master 330 hasan upper surface 332 including thereon positive relief topologicalfeatures 334 protruding above surface 332 that are shaped, sized, andpositioned to form channels within the molded replica structurecorresponding to a desired arrangement of connecting channels within thethird, intermediate level of the microfluidic network structure beingfabricated. In addition, if desired, additional features (not shown) canbe included on the surface 332 of mold master 330 corresponding tochannels that are disposed within (i.e., have longitudinal axis coplanarwith) the third, intermediate level of the microfluidic networkstructure being formed.

[0097] Step 7 involves curing PDMS prepolymer 310 (or other hardenableliquid) as previously described for Step 2 above, and Step 8 involvesselectively removing molded replica layer 340 from mold master 330 whileit remains supported by an adherent to upper mold master 306, asdescribed for Step 3 above. Optional step 9 involves removing moldedreplica layer 340 from upper mold master 306 and, if desired, trimminglayer 340 so that it has an essentially identical overall size andperimeter shape as layer 318 above. Step 10 involves placing moldedreplica layer 340 into conformal contact with upper surface 317 ofmolded replica layer 318, aligning the channels 342 in molded replicalayer 340 with channels 321 in molded replica layer 318 to provide adesired registration between the channels of the first, lower level ofthe structure comprised of layer 318 and the third, intermediate levelof the structure comprised of layer 340, followed by irreversiblysealing together layers 318 and 340. In alternative embodiments, thealignment and sealing steps can be delayed if desired and performed inone step for all of the layers (i.e., all three channel-forming layers)comprising the overall structure which have been formed and stacked uponeach other (e.g. see FIG. 5c below). In addition, for embodimentswherein upper mold master 306 is transparent, for example forembodiments where upper mold master comprises PDMS, and especially forembodiments including replica layers having a large number of channelsdisposed completely through the entire thickness of the membrane layeror having channels shaped so that the molded replica membrane layer isnot free-standing when removed from a support surface (e.g., channelscomprising continuous, closed geometric shapes, spiral shaped channels,etc.), layer 340 is preferably not removed from mold master 306 asillustrated in Step 9, but instead, mold master 306, with molded replicalayer 340 attached thereto, is placed in contact with upper surface 317of molded replica layer 318 and aligned and sealed as described in step10 prior to removing mold master 306, so that the molded replica layerremains attached to and supported by a mold master during each of themanipulation steps and is never free-standing.

[0098] Alignment of the molded replica features comprising the channelsof layers 318 and 340 can be accomplished utilizing a microscope, suchas a stereo microscope, in combination with an alignment stage and/ormicromanipulators for accurately positioning the layers and registeringthe features with respect to each other. For a preferred embodimentwherein layers 318 and 340 are comprised of PDMS, layers 318 and 340 canbe aligned and sealed to each other by either of the preferred methodsdescribed directly below. In a first method, layer 340 is placed uponlayer 318 and carefully aligned with respect to layer 318 to provide adesired alignment and registration of channels by utilizing a stereomicroscope and a micromanipulator. Layers 318 and 340 are then carefullyslightly separated from each other (e.g. by a few millimeters), withoutchanging the registered lateral alignment of channels within the layers,to provide a small space between surface 317 of layer 318 and surface341 of layer 340. The aligned structure having the layers slightlyseparated is then exposed to an oxygen-containing plasma in order tooxidize surfaces 317 and 341. The layers are then carefully broughttogether without altering or disturbing the lateral alignment of thechannels, so that surfaces 317 and 341 spontaneously seal to each otherupon conformal contact.

[0099] In the second, especially preferred, embodiment, alignment andsealing of the layers proceeds as follows. The upper surface 317 oflayer 318 and lower surface 341 of layer 340 are oxidized utilizing theoxygen-containing plasma exposure method described previously, and aliquid that is essentially non-reactive with the oxidized surfaces isplaced upon layer 317 to form a continuous layer of liquid thereupon,upon which, surface 341 of layer 340 is placed. The liquid, in additionto being essentially non-reactive with the oxidized surfaces of thePDMS, also preferably prevents degradation of the active Si—OH groupspresent on the surfaces for a period of time sufficiently long to enablealignment of the surfaces with respect to each other and removal of theliquid. After placing layer 340 onto the fluid-covered surface of layer318, layer 340 is aligned with respect to layer 318 to yield a desiredregistration and alignment of features (channels) for forming themicrofluidic network structure. The non-reactive liquid is then removedfrom between the two surfaces bringing the two surfaces into conformalcontact with each other and spontaneously sealing the two surfacestogether.

[0100] A variety of liquids can potentially be utilized as thenon-reactive liquid in the context of the inventive alignment methodabove described. As previously discussed, appropriate liquids will beessentially non-reactive with the oxidized surfaces and will preferablystabilize and delay degradation of the active chemical groups containedwithin the oxidized surfaces. It has been found, in the context of thepresent invention, that polar liquids, and especially those comprisingcompounds including hydroxyl moieties, are effective for use as thenon-reactive liquid. Especially preferred are water, alcohols, andmixtures thereof with alcohols, and alcohol-water mixtures beingparticularly preferred, especially those including methanol and/ortrifluoroethanol. The non-reactive liquid, in preferred embodiments, isremoved from between the oxidized surfaces of the layers via evaporationof the liquid, and thus, in such embodiments, as the non-reactive liquidevaporates the oxidized surfaces of the layers are simultaneouslybrought together in conformal contact whereupon the surfaces react tocreate an essentially irreversible seal.

[0101] While we have described above an embodiment wherein layer 340comprising the third, intermediate layer of the structure is aligned andsealed with respect to layer 318 comprising a first, lower level of thestructure prior to the fabrication of the molded replica layercomprising a second, upper level of the structure, in other embodiments,as mentioned above, the upper layer is formed prior to sealing the lowerand intermediate layers together, so that the intermediate and upperlayers can be stacked, aligned, and sealed to the lower layer in asingle step, eliminating the need to selectively oxidize only lowersurface 341 of intermediate layer 340 so as to prevent degradation of anoxidized upper surface 343 of intermediate layer 340 prior to theformation, stacking, and alignment of the upper layer to theintermediate layer (as shown and described in FIG. 5c below).

[0102]FIG. 5c illustrates the final steps for forming the overallthree-layer, three-level microfluidic network according to this firstfabrication method embodiment of the invention. Step 11 and Step 12 ofFIG. 5c are analogous to Steps 1 and 2 of FIG. 5a and Steps 6 and 7 ofFIG. 5b and involve sandwiching a hardenable liquid 310, such as PDMS,between upper mold master 306 and a lower mold master 350 having anupper surface 352 including thereon topological features 354 in positiverelief constructed and positioned for forming channels disposed withinthe second, upper level of the final overall microfluidic networkstructure. Hardenable liquid 310 is cured and solidified in Step 12, aspreviously described, and, in preferred embodiments, molded replicalayer 360 is preferentially separated from surface 352 of lower moldmaster 350 while remaining in contact with and supported by upper moldmaster 306, as previously described. Molded replica layer 360, whichcomprises the second, upper level of the overall structure, includesmolded channels 362 disposed within layer 360. Step 14 involvesoptionally removing molded replica membrane layer 360 from upper moldmaster 306, as previously described for Step 9 discussed in the contextof FIG. 5b. In step 15, molded replica layer 360, formed in Step 12above, is stacked upon intermediate layer 340, produced as described inthe context of FIG. 5b above, and is subsequently aligned with respectto lower layers 340 and 318 such that channels 362 are registered andarranged in a desired alignment with respect to channels 342 of layer340 and channels 321 of layer 318 to provide a desired overallthree-dimensional fluidic network structure. Layer 360 is preferablysealed to layer 340 by utilizing one of the aligning and sealing methodspreviously described in the context of Step 10 of FIG. 5b above.

[0103] As previously mentioned, in some preferred embodiments, layer 340is aligned with respect to layer 318 and layer 360 is aligned withrespect to layer 340 and the layers are sealed together in a single stepafter alignment, which step, for such embodiments, can take place atStep 15 of FIG. 5c. In such embodiments, layer 340 would not beirreversibly sealed to layer 318 prior to the addition of layer 360 tothe stack and alignment of layer 360 with respect to layer 340 and 318.In such embodiments, wherein layers 340 and 360 are both aligned andsealed in a single step, the alignment and sealing methods utilized canbe essentially the same as those previously described for aligning andsealing layer 340 to layer 318 in the context of Step 10 of FIG. 5b. Inaddition, in some embodiments where it is desired to irreversibly sealtogether some portions of the surfaces of the layers of the structureswhile leaving non-irreversibly sealed other portions, such portionswhich are not desired to be irreversibly sealed can be coated with aprotective coating (e.g. petroleum jelly) prior to oxidation in order toprevent oxidation of that portion of the surface so that it will notirreversibly seal to other oxidized surfaces upon contact.

[0104] Also provided, according to the invention, is a method forself-aligning layers 318, 340, and 360 with respect to each other toprovide a desired alignment and registration of the channels within eachof the layers, without the need for manual alignment with the aid of amicroscope and/or micromanipulator. The self-alignment method providedaccording to the invention can be utilized for the embodiments describedabove wherein the layers are oxidized and separated from each other by alayer of liquid during alignment of the layers. Details of thisself-alignment method are described below in the context of FIG. 6 andrely on the interaction between the surface tension of the liquidbetween the layers and specific alignment features provided within thelayers being aligned.

[0105] Microfluidic network structure 370 obtained at the conclusion ofStep 15 of FIG. 5c can comprise, for some embodiments, a completestructure, useful, for example, for applications wherein it is desiredthat channels 362 in layer 360 remain uncovered and exposed to thesurroundings. For example, one particular embodiment utilizing amicrofluidic network structure similar in configuration to structure 370involves utilizing the microfluidic network structure as a stampingtemplate for selectively applying a fluid to a material surface tocreate a pattern on the material's surface corresponding to the patternof channels 362 in layer 360. In such embodiments, surface 364 of layer360 comprises a stamping surface, which is placeable in contact with amaterial surface for forming a pattern thereon, and microfluidic networkstructure 370 comprises a three-dimensional microfluidic stamp. Specificuses and patterns producible by such microfluidic stamps are describedin greater detail below.

[0106] For other embodiments where it is desired to form a microfluidicnetwork structure having an enclosed network of channels, optional Step16 of FIG. 5c involves contacting upper surface 364 of layer 360 with anupper substrate layer 380 to form enclosed microfluidic networkstructure 390. In some preferred embodiments, where layers 318, 360, and364 comprise PDMS, upper substrate layer 380 is also comprised of PDMSand is irreversibly sealed to surface 364 via the self-sealing methodutilizing oxidation of the PDMS surfaces with an oxygen-containingplasma described in detail above. In alternative embodiments, however,upper substrate layer 380 may simply be placed in conformal contact withupper layer 364 and not irreversibly sealed thereto. In addition, uppersubstrate 380, in some embodiments, is not formed of the same material(e.g., PDMS) as layers 318, 360, and 364 of the structure. Uppersubstrate 380 can be essentially any of the materials mentionedpreviously for comprising substrate layer 320 previously described abovein the context of FIG. 5a or any other substrate which can contactsurface 364 conformally.

[0107] In order to provide fluid communication between channelscontained within layers 318, 360, and 364 of structure 390 and thesurrounding environment, lower substrate layer 320 and/or uppersubstrate layer 380 can include, formed therein, inlet/outlet conduits392 providing fluid communication between the channels of the structureand the external environment. Conduits 392 can be formed withinsubstrate layer by a variety of machining and/or molding methods, aswould be apparent to those of ordinary skill in the art. In oneembodiment, the conduits 392 in substrate 320, comprising PDMS, areformed by carefully boring into layer 320 with a small diameter syringeneedle. In other embodiments, substrate layer 392 can itself comprise areplica molded structure with conduits 392 corresponding to and formedby topological features present on a surface of a mold master utilizedto form substrate layer 320. In addition, as would be apparent to thoseof ordinary skill in the art, other features can be machined within, ormolded within one or both of substrate layers 320 and 380 to providevarious desired structures and functions for particular applications.For example, upper substrate layer 380 as shown includes traversingtherethrough a small diameter channel 394, having a characteristic crosssectional dimension on the order of a few microns to a few tens ofmicrons, which conduit 394 serves the function of providing a reliefvalve to prevent over pressure of the channels contained within thestructure defined by layers 318, 340, and 360.

[0108]FIGS. 6a-6 c illustrate one method for self-aligning variouslayers of the microfluidic network structures with respect to each otherprovided by the invention. The self-alignment method outlined in FIGS.6a-6 c can be utilized for embodiments involving the alignment andsealing methods discussed above involving disposing layers of thestructure separated from each other by a layer of liquid disposedtherebetween. Such a method is useful, for example, for aligning layers340 and 318 with respect to each other and layers 360 and 340 withrespect to each other in the previously described microfluidic networkfabrication method. In addition, the self-alignment method described inFIGS. 6a-6 c can also be utilized for performing self-alignment in thecontext of the methods described below in FIG. 7 and FIG. 10.

[0109] One embodiment for implementing the self-aligning method providedaccording to the invention is illustrated in FIG. 6a. FIG. 6a shows afirst layer 400 and a second layer 402 including therein replica moldedfeatures 404 and 406 respectively, comprising, for example, channelsdisposed within each of the layers, which channels are desired to beregistered and aligned with respect to each other in a certain way. Inthe illustrated embodiment, a plurality of self-alignment elements 408are formed at selected, predetermined locations within layer 400 andlayer 402. In the illustrated embodiment, self-alignment features 408comprise vertically disposed channels traversing, in some preferredembodiments, essentially completely through layers 400 and 402 such thatupon bringing layer 402 into conformal contact with layer 400 uppersurface 410 of layer 402 is in fluid communication with lower surface412 of layer 400 through vertically disposed channels formed by thealignment of the self-alignment elements contained within layers 400 and402 respectively. In other embodiments, one or more of the alignmentelements may not completely traverse the layer in which it is disposed,but may instead comprise an indentation, bump, or other feature withinor on the surface of the layer.

[0110] In order to effect proper self-alignment, it is important thatlayers 400 and 402 be essentially identical in size and perimetricshape, when viewed in the x-y plane along the negative z-axis directionas illustrated, such that the perimeter of layers 402 and 400essentially identically overlap when the layers are brought togetherinto properly aligned conformal contact. Optionally, in otherembodiments, proper self-alignment can also be effected if, instead ofbeing essentially identical in size and perimetric shape, one of thelayers is much larger than the other so that the meniscus of liquidformed around the edge of the smaller layer does not change appreciablyin total surface area with small movements of the two layers withrespect to each other.

[0111] Self-alignment elements 408, in preferred embodiments, are formedwithin layers 400 and 402, during the replica molding process forforming the layers, by topological features provided within the moldmasters utilized for molding. Such topological features can bepositioned and located within the mold master surface at selected,strategic positions with respect to features within the mold mastersurface for forming channels 404 and 406 through use of a CAD computerprogram, such as described above for designing the overall layout of thetopological features for forming the various channels within the replicamolded layer structures. Topological features forming self-alignmentelements 408 are positioned with respect to topological features formingchannel structures 404 and 406 so that when layer 400 and layer 402 aresuperimposed such that alignment holes 408 are precisely aligned withrespect to each other, channel features 404 and 406 are also orientedwith respect to each other in a desired registered alignment.

[0112]FIG. 6b and FIG. 6c illustrate the manner by which alignment holes408 interact with a fluid layer 412 disposed between layers 400 and 402to effect self-alignment. When self-alignment holes 408 and features 404and 406 are properly aligned with respect to each other, as shown inFIG. 6b, the layers are arranged in an equilibrium position in which theinterfacial area 414 between fluid layer 412 and the surrounding gaseousenvironment is minimized and there are no net capillary forces, due tothe surface tension of fluid layer 412, tending to change the positionof layer 400 or layer 402 with respect to each other.

[0113] By contrast, when features 404, 406, and self-alignment holes 408are misaligned with respect to each other, as illustrated in FIG. 6c,the interfacial area 414 between fluid layer 412 and the surroundinggaseous atmosphere is increased with respect to the interfacial surfacearea when the system is in its equilibrium position as shown in FIG. 6babove, and there will be a net resulting capillary force in thedirection shown by arrow 416, due to surface tension effects of fluidlayer 412, tending to bring the system into the equilibrium positionillustrated in FIG. 6b.

[0114] In alternative embodiments, an essentially identicalself-aligning effect as illustrated in FIGS. 6a-6 c can be achievedwithout the need for forming self-alignment holes or features, such as408, in the layers which are to be self-aligned with respect to eachother. In such alternative embodiments, the layers can be formed withoutself-alignment holes, such as 408, but instead be formed or trimmed tohave perimeter shapes, which are essentially identical to each other, sothat the layers when stacked upon one another with a fluid layertherebetween, as illustrated in FIGS. 6b and 6 c, will have a minimumfree energy equilibrium position defined by an essentially precise andexact overlay of the essentially identical perimetric shapes of the twolayers. The features comprising channels within the layers are, in suchembodiments, strategically positioned with respect to the peripheralborder of the layers, so that, when the layers are aligned in theabove-described minimum energy, no net capillary force equilibriumposition, the perimeters of the layers are precisely superimposed uponeach other and the features comprising the channels within the layersare also similarly aligned with respect to each other in a desiredregistration. FIG. 6d illustrates one contemplated embodiment of aperimetric shape for enabling the above-described self-alignment ofvarious layers of the structure without the need for alignment holes.

[0115] The above-described self-alignment techniques are able toself-align a stack of as many individual layers as is desired,essentially simultaneously and in parallel. The self-alignment techniquedescribed herein is also capable of self-aligning elements with respectto each other within a margin of error of approximately +/−10 μm orless, providing sufficient alignment precision for most of the channelsizes and configurations contemplated for the structures providedaccording to the invention (e.g., channel structures having across-sectional dimension ranging from about 20 μm to about 500 μm). Thealignment precision obtainable by the above-described self-alignmenttechnique is typically comparable or better than that obtainable viamanual alignment techniques utilizing a stereomicroscope andconventional micromanipulation equipment.

[0116] The above-described self-alignment techniques are especially wellsuited for embodiments involving alignment of oxidized PDMS layersutilizing the above-described alignment/sealing method using anon-reactive liquid disposed between and able to wet the oxidized PDMSlayers. However, those of ordinary skill in the art will readilyrealized that the above-described self-alignment technique can beutilized for aligning layers comprised of essentially any of thesuitable materials for forming the microfluidic system discussed aboveand can be utilized for self-aligning layers that are not reactive withrespect to each other and do not become essentially irreversibly sealedto each other upon contact but, instead, are simply aligned inconformal, non-sealing contact with each other. Those of ordinary skillin the art can readily select appropriate liquids having desiredsurface-wetting properties (for use in the self-aligning technique whenutilizing the technique to self align surfaces comprised of materialsother than oxidized PDMS) using no more than known, published surfacewetting properties for various liquids on various surfaces or routinescreening tests not requiring undue experimentation. In addition, whilethe above-described self-alignment technique has been exemplified in thecontext of aligning two replica molded layers of the overallmicrofluidic structure with respect to each other. In other embodiments,the technique can be utilized to-align a replica molded layer comprisingone or more levels of the microfluidic structure to the surface of asubstrate, for example a silicon microchip, or the like. Utilization ofthe self-aligning method for aligning a layer of the microfluidicnetwork to a substrate surface, for example a surface of a siliconmicrochip, may be important for applications where the microfluidicnetwork is utilized as an on-chip sensor, detector, analyzer, etc.

[0117]FIG. 7 illustrates an alternative embodiment for fabricating amicrofluidic network structure according to the invention. Unlike themethod previously described in the context of FIGS. 5a-5 c, thefabrication method described in FIG. 7 involves the formation, byreplica molding, alignment, and assembly of only two, as opposed tothree, discrete layers forming the three levels of the overallmicrofluidic network structure.

[0118] As described above in the context of FIGS. 5a-5 c, the methodoutlined in FIG. 7 can potentially utilize a wide variety of hardenableliquids for forming the replica molded components of the microfluidicnetwork structure. Such hardenable liquids were described previously inthe context of FIGS. 5a-5 c. As previously, in preferred embodiments,the replica molded structure is formed of a polymeric material, morepreferably an elastomeric material, and most preferably a transparentelastomeric material. In a particularly preferred embodiment illustratedand exemplified in FIG. 7, the replica molded structures are formed ofPDMS.

[0119] In Step 1 of the method illustrated in FIG. 7, a mold master 500having a surface 502 including a series of topological features 504thereon protruding from the surface in positive relief is formed in amanner essentially equivalent to that described for forming mold master300 of FIG. 5a. Topological features 504 are shaped, sized, and laid outon surface 502 in a pattern predetermined to form a desired arrangementof channels disposed in the upper, third level of the overallmicrofluidic network structure. Mold master 502 is then placed in thebottom of a petri dish or other container having a depth exceeding theheight of the upper surfaces of topological features 504 on surface 502.

[0120] In Step 2, a hardenable liquid is added to the containercontaining master 500 in an amount sufficient to completely cover andsubmerge topological features 504. As discussed in FIG. 5a above,surface 502 of mold master 500, in preferred embodiments, is treatedwith a release agent, for example a silanizing agent, to permit releaseof the replica molded structure from the surface without undue damage ordistortion of the replica molded structure. Also in Step 2, as describedabove in the context of FIGS. 5a-5 c, the hardenable liquid, for examplea PDMS prepolymer solution, is cured and solidified to form a solidmolded replica 510 of surface 502 of mold master 500. Molded replica 510is removed from surface 502 after curing as illustrated in Step 2. Inthe illustrated embodiment, molded replica 510 comprises a PDMS slabwhich can, as illustrated, be trimmed to a desired overall size andperimetric shape. Molded replica 510 includes therein, but notcompletely extending therethrough, a series of indentations 512 in lowersurface 514 corresponding to topological features 504 of mold master500. Indentations 512 form channels disposed within the third, upperlevel of the overall microfluidic network to be fabricated.

[0121] Steps 3 and 4 of the method illustrated in FIG. 7 comprise theformation of a replica molded membrane layer including therein bothchannels disposed in the first, lower level of the overall microfluidicnetwork structure and connecting channels of the third, intermediatelevel of the overall microfluidic network structure forming fluidicconnections between the channels disposed in the first, lower level andthe second, upper level of the structure. The molded replica membranelayer, having two levels of features formed therein, is formed by amembrane sandwich fabrication method (Steps 3 and 4) similar to themethod previously described in the context of FIGS. 5a-5 c, except thatmold master 520 includes a surface 522 having formed thereon a pluralityof topological features 524 in positive relief protruding from surface522, that include features, for example feature 526, that are two-leveltopological features, which are characterized by a first portion 528having a first height with respect to a region of surface 522 adjacentto feature 526 and a second portion 530, which is integrally connectedto the first portion, having a second height with respect to surface 522adjacent feature 526, which second height is greater than the height offirst portion 528.

[0122] The term “integrally connected,” as used herein in the context ofdescribing two-level topological features of mold masters, refers tosuch features having at least a first portion and a second portion, thesecond portion having a height or depth with respect to the surface ofthe mold master adjacent the feature different from the first portion,wherein the first and second portion comprise two different regions of acontinuous structure or comprise two discrete structures each having atleast one surface in direct contact with at least one surface of theother. By providing such two-level topological features on mold master520, the illustrated method allows simultaneous formation and alignmentof channels disposed within two levels of the overall microfluidicnetwork structure. Thus, by forming two levels of the overall structurewithin a single layer in a single replica molding step, the presentmethod eliminates the need to align two discrete layers comprising thefirst, lower level of the -structure and the third, intermediated levelof the structure with respect to each other after formation of themolded replica layers. Thus, as described below, the present methodrequires only a single alignment step for assembling the molded replicalayers into the overall microfluidic network structure.

[0123] A variety of photolithography and micromachining methods known tothose of ordinary skill in the art, which are capable of formingfeatures on a surface having multiple heights or depths with respect tothe surface, can potentially be utilized in the context of the presentinvention for forming the two-level topological features 526 of moldmaster 520. A particularly preferred embodiment for forming mold master520 involves an inventive method for forming two-level topologicalfeatures in photoresist, and is described in more detail below in thecontext of FIG. 8.

[0124] After formation of mold master 520, a layer of hardenable liquid,for example PDMS, is placed upon surface 522 of mold master 520 andcovered with an upper mold master 540, having a lower surface 542 thatis essentially flat and featureless, so that surface 542 is in conformalcontact with the uppermost surfaces of the two-level topologicalfeatures 526 on surface 522 of mold master 520. As previously describedin the context of FIGS. 5a-5 c, mold master 540 can comprise a varietyof materials including, for example, an elastomeric polymer slab, forexample formed of PDMS, a polymeric sheet, a flat silicon wafer, etc. Inpreferred embodiments, as previously discussed, it is desirable that theinterfacial adhesion strength between surface 522 of mold master 520 andthe hardened molded replica differ from the interfacial surface adhesionbetween surface 542 of mold master 540 and the hardened liquidcomprising the molded replica. In the illustrated embodiment, surface522 of mold master 520 comprises a silanized polymeric negativephotoresist layer and mold master 540 comprises a Teflon™ (PTFE) sheet.

[0125] In Step 4, pressure is uniformly applied to surface 544 of uppermold master 540 and surface 546 of lower mold master 520 to enable theupper surfaces 548 of topological features 526 to make sealing contactwith surface 542 of mold master 540 during the hardening and curingprocess forming the replica molded membrane layer. In Step 4, thehardenable liquid, for example PDMS prepolymer, is cured to form atwo-level replica molded membrane 550. Two-level replica molded membrane550 includes a plurality of first channels 552, disposed within a lowersurface 554 of the membrane, comprising channels disposed within thefirst level of the overall microfluidic network structure, and alsoincludes vertically oriented connecting channels 554 that completelypenetrate the thickness of the membrane and interconnect lower surface554 and upper surface 556 of the membrane, forming the connectingchannels disposed within the third, intermediate level of the overallmicrofluidic network structure. Channels 552 comprise replica moldedfeatures corresponding to first portions 528 of topological features 526of mold master 520 and connecting channels 555 comprise replica moldedfeatures corresponding to second portions 530 of two-level topologicalfeatures 526 of mold master 520.

[0126] In the illustrated embodiment, the PDMS membrane comprisingmolded replica layer 550 is separated from the mold masters by firstpeeling PTFE sheet 540 from the upper surface 556 of the membrane andsubsequently peeling the membrane from upper surface 522 of mold master520. In other embodiments, molded replica 550 can remain in contact withupper surface 522 of mold master 520 during the subsequent, and belowdescribed, aligning and sealing steps, in order to support membrane 550and prevent distortion or destruction of the molded features therein. Itshould be understood, that for more complex structures, additionalreplica molded membranes such as 550 can be stacked upon each other inthe assembly of the microfluidic network structure to yield structureshaving more than three levels of interconnected microfluidic channels.

[0127] In the final step of fabrication, Step 5, replica molded slab 510and replica molded membrane 550 are aligned with respect to each otherto yield the desired microfluidic network structure, brought intoconformal contact with each other, and optionally sealed together bymethods previously described above in the context of FIGS. 5a-5 c toyield the final microfluidic network structure 560. As previouslydescribed, the structure 560 can include inlet conduits 562 and outletconduits 564 for each of the non-interconnected fluid flow pathsdisposed within the structure, or other interconnections between theflow paths within the structure and the external environment as requiredor desired for a particular application. In the illustrated embodiment,microfluidic network structure 560 includes three non-fluidicallyinterconnected fluid flow paths therein. The first flow path 561 has aninlet and outlet in the foreground and is shaded light gray; the secondflow path 563 has an inlet and outlet that are centrally disposed shadedin black; and the third flow path 565 has an inlet and outlet in thebackground and is shaded dark gray.

[0128] In addition, lowermost surface 554 of structure 560 includestherein a pattern indentations corresponding to the channels of thefirst, lower level of the microfluidic network structure formed withinthe bottom surface 554 of the replica molded membrane 550. Thus,microfluidic network structure 560 is useful for embodiments wherein themicrofluidic network structure is utilized as a surface patterning stampfor depositing materials onto a material surface in a patterncorresponding to the channels disposed within surface 554, or otherwisecreating a patterned surface with a pattern corresponding to the patternof the channels disposed within surface 554. In alternative embodiments,surface 554 can be placed in conformal contact with, and optionallysealed to, a solid PDMS slab, or other substrate or surface, to form anenclosed microfluidic network structure, as described previously in thecontext of FIGS. 5a-5 c.

[0129]FIG. 8 illustrates a preferred method for preparing mold mastersthat have a surface including thereon one or more two-level topologicalfeatures. While the illustrated method is useful for forming two-leveltopological features in layers of either negative or positivephotoresist materials, in the embodiment illustrated, a negativephotoresist material (e.g., SU8-50) is utilized as an example. Inaddition, while, in the illustrated embodiment, two-level topologicalfeatures comprising positive, high-relief features protruding from thesurface of the mold master are fabrinated, it should be understood thatthe method is also well suited to produce two-level topological featurescomprising negative, low-relief features characterized by indentations,grooves, or channels within the surface of the mold master. Anyvariations in the below described technique for producing two-levelpositive, high-relief features in negative photoresist that are requiredin order to produce two-level features in positive photoresist and/or toproduce two-level features comprising negative, low-relief featuresinvolve only simple extensions of the below-described method that wouldbe apparent to those of ordinary skill in the art.

[0130] In Step 1 of the method illustrated in FIG. 8, a silicon wafer600, or other suitable substrate, is coated with a layer of photoresist602, by a conventional spin-coating technique or other suitable coatingtechnique known to those of ordinary skill in the art. Layer 602 isspin-coated to a depth corresponding to the desired depth of the deepestfeature to be formed on the first level of the mold master (e.g. a depthcorresponding to the deepest channel to be disposed in the level of themicro fluidic channel structure to be replica molded by the first Ievelof the mold master. The thickness of layer 602 will typically range fromabout 20 μm to about 500 μm, and can, in some embodiments, be as thickas about 1 mm.

[0131] In Step 2, the photoresist is “soft baked” by being exposed to anelevated temperature for a short period of time to drive off solventused in the spin-coating process For example, for SU8-50 negativephotoresist, the coated substrate is exposed to a temperature of about95-105° C. for a period of several-minutes. In Step 3, a-first photomask604 including thereon a pattern 606 corresponding to features 626 of thefirst level of the mold master is placed in contact with negativephotoresist layer 602. As would be apparent to those of ordinary skillin the art, a wide variety of photomasks can be utilized according tothe present inventive method; however, in a preferred embodimentillustrated, photomask 604 comprises a high resolution transparency filmhaving a pattern printed thereon. Designs for the channel system printedupon the high resolution transparency are preferably generated with aCAD computer program. In the illustrated embodiment, a high-resolution(e.g., 3000-5000 dpi) transparency, which acts as photomask 604, isproduced by a commercial printer from the CAD program design file. Inthe illustrated embodiment, essentially the entirety of photomask 604 isrendered opaque to the radiation used to expose the photoresist by alayer of toner, and the fluidic channel-forming features to be formed onthe surface of negative photoresist 602 correspond to transparentregions 606 of the photomask surface.

[0132] In addition to regions 606 corresponding to features in the moldmaster for formning fluidic channels within the molded replica structureformed with the mold master, photomask 604 also includes peripheraltransparent regions 608, which correspond to topological features forforming alignment tracks useful for aligning the mold masters withrespect to each other in certain methods for forming microfluidicstructures as described in more detail below in FIGS. 9a and 9 b.

[0133] In Step 4, upper surface 603 of photoresist layer 602 is exposedto radiation, for example ultraviolet (UJV) radiation of a frequency andintensity selected to cross-link exposed areas of the negativephotoresist, through the transparent regions of the printed pattern ofphotomask 604. In Step 5, after exposure to cross-linking radiation, thefirst photomask 604 is removed from the surface, the photoresist ishard-baked (e.g. at about 95-105° C. for several minutes) and a secondlayer of photoresist is spin-coated on top of surface 603 of photoresist602. The second layer of photoresist is spin-coated to a thicknesssufficient for forming features in the mold master corresponding to theconnecting channels disposed within the third, intermediate level of thereplica molded microfluidic network structure formed with the moldmaster. Typically, the thickness of the second level of photoresist willrange from about 20 μm to about 1 mm. Wafer 600, now containing a first,exposed layer of photoresist and a second layer of unexposed photoresistcan then be subject to another soft-baked procedure to drive off solventfrom the unexposed layer of photoresist, similarly as described in Step2 above.

[0134] As illustrated in Step 5 of FIG. 8, regions of the first layer ofphotoresist that were exposed to the radiation (e.g., regions 610 and612) typically exhibit a change in the degree of transparency and/orrefractive index of the photoresist, thus rendering them visible throughthe upper layer of newly spin-cast, unexposed photoresist. Thisvisibility allows a second photomask to be easily aligned with respectto the first exposed pattern by using a standard photomask aligner. Inother embodiments, especially where the exposed pattern may not bevisually apparent, visible alignment features or elements can beincluded on the surface of wafer 600 to enable alignment of the secondphotomask to achieve a desired two-level pattern, as would be apparentto those of ordinary skill in the art.

[0135] In Step 6, a second photomask 614 including thereon printedpatterns 616, corresponding the second level portions of the two-leveltopological features of the mold master, which form the connectingchannels in the intermediate level of the replica molded microfluidicnetwork structure formed with the mold master, and 618, corresponding toa second level of the optional alignment tracks. It should beunderstood, that while, in the illustrated embodiment, features 606corresponding to topological features for forming channels disposed inthe first level of the microfluidic network structure comprise linearfeatures, in other embodiments, features 606 can be non-linear, thusforming curved topological features resulting in non-linear, curvedchannels within the first level of the microfluidic structure.Similarly, any of the previously described structures and methods forforming channels disposed within a particular level of microfluidicnetwork structure can include channels that are non-linear and curvedwithin the plane or curved surface defining the level of themicrofluidic network structure in which the channels are disposed inaddition to, or instead of, the straight channels previouslyillustrated.

[0136] Printed pattern 616, creating topological features for formingchannels within the microfluidic network structure can also, in someembodiments, include features parallel and contiguous with regions 610formed within the first layer of photoresist and corresponding toprinted pattern 606, such that some of the topological features producedon the surface of the mold master by the illustrated method includefeatures that form channels having a longitudinal axis parallel to thefirst level of the replica molded microfluidic network structure formedwith the mold master, and which have an overall depth within the replicamolded microfluidic network structure formed with the mold master, whichis equal to the combined depth of the first level and the third,intermediate level of the structure (i.e., for forming replica moldedmicrofluidic network structures having deep channels that are disposedwithin both the first level and the third, intermediate level of themicrofluidic network structure).

[0137] Photomask 614 is aligned in Step 6 with respect to exposedpattern 610 and the second, unexposed layer of photoresist is exposed,in Step 7, to the cross-linking radiation through photomask 614.Following exposure, mask 614 is removed from the top layer ofphotoresist, and the photoresist is hard-baked as described above. Ifdesired, the above-mentioned steps can be repeated with additionallayers of photoresist and additional photomasks to produce more than twolevels of topological features on the surface of wafer 600. After thedesired number of layers of photoresist have been coated onto wafer 600and exposed to cross-linking radiation as described above, the reliefpattern is developed in Step 8 by exposing the photoresist to adeveloping agent that dissolves and removes non-crosslinked photoresistmaterial leaving behind a mold master 620 having a surface 622 includingthereon a pattern of two-level high relief features 624 having a firstportion 626 with a first height above surface 622 and a second portion628 having a second height above surface 622, which is greater thanheight 626. First portion 626 of the topological features forms thechannels disposed within the first level of the replica moldedmicrofluidic network structure formed with mold master 620, and secondportion 628 of the topological features forms the connecting channelstraversing the third, intermediate level of a microfluidic networkstructure replica molded using mold master 620.

[0138] Also formed on surface 622 of mold master 620 by theabove-outlined process are alignment tracks 630 having a heightcorresponding to the height of the second portion 628 of topologicalfeatures 624. While, in the illustrated embodiment, the second layer ofphotoresist was spin-coated onto a first layer of exposed photoresistbefore developing the first layer, in an alternative embodiment, thefirst layer of photoresist can be developed before spin-coating thesecond layer of photoresist if desired. Solvents useful for developingthe unexposed portions of the photoresist are selected based on theparticular photoresist material employed. Such developing agents arewell known to those of ordinary skill in the art and are typicallyspecified by the commercial manufacturers of many of the photoresistsuseful for performing the methods of the invention. For example, for theillustrated embodiment utilizing SU8-50 negative photoresist,uncross-linked photoresist can be removed during development by exposingthe photoresist to propylene glycol methyl ether acetate. Two-level moldmaster 620, subsequent to formation as described above, is preferablycoated with a release agent, for example by silanizing the surface, inorder to facilitate removal of a molded replica from the surface of themold master.

[0139]FIGS. 9a and 9 b illustrate the steps of a third embodiment of themethod according to the invention for fabricating a three-dimensionalmicrofluidic network structure. The method illustrated in FIGS. 9a and 9b comprises a membrane sandwich technique similar to that previouslydescribed in Steps 3 and 4 of the method illustrated in FIG. 7, exceptthat instead of forming a replica molded membrane layer between a bottommaster including two-level topological features and a top mold masterhaving an essentially flat, planar surface, as was illustrated in themethod of FIG. 7, in the method according to FIGS. 9a and 9 b, a replicamolded membrane layer is formed between two mold masters, both includingtopological features thereon and at least one including at least onetwo-level topological feature thereon, thus yielding a replica moldedmembrane including therein a microfluidic network structure containingall three of the above-discussed levels. In some embodiments, both theupper and lower mold masters utilized for forming the three-levelreplica molded membrane layer according to the embodiment of FIGS. 9aand 9 b can comprise mold masters, for example similar to mold masters500 and 520 shown in FIG. 7. However, as previously discussed, it isdesirable for at least one of the mold masters to be formed of anelastomeric material to improve sealing contact between portions of thesurfaces of the mold masters that are in contact during the replicamolding process so as to prevent undesirable leakage of hardenableliquid into such regions of contact. Therefore, in preferredembodiments, the upper mold master and/or lower mold master are formedfrom an elastomeric material having a surface with topological featuresthereon.

[0140] In some particularly preferred embodiments, elastomeric moldmasters are formed using a replica molding procedure, similar to thatused to form the various layers of the microfluidic structure, to formtopological features on the elastomeric mold master that are formedduring replica molding from topological features on a pre-masterprepared by photolithography or micromachining. The method illustratedin FIGS. 9a and 9 b correspond to such a preferred embodiment. In theillustrated embodiment, the top mold master, as well as the replicamolded membrane layer, are formed from an elastomeric materialcomprising PDMS. As referred to and discussed extensively above, PDMS,while being preferred for forming many of the structures and moldmasters according to the invention, comprises only one example of amaterial formable from a hardenable liquid useful for forming the moldmasters and microfluidic networks according to the invention. A widevariety of alternative materials and hardenable liquids have beenpreviously discussed in the context of the methods illustrated in FIGS.5 and 7, and such materials, or other materials apparent to those ofordinary skill in the art, can be substituted for PDMS in the methodillustrated in FIGS. 9a and 9 b below.

[0141]FIG. 9a illustrates one preferred method for forming anelastomeric top mold master for use in forming a three-level replicamolded membrane layer. In Step 1, a pre-master mold is fabricated byforming topological features on a surface of a substrate 700, forexample as previously illustrated in the context of FIG. 8. Since, inthe illustrated embodiment, it is desired that the topological featuresformed in the replica molded top mold master comprise positive,high-level relief features protruding from the surface of the moldmaster, the topological features formed on surface 702 of substrate 700comprise negative, low-level relief features characterized by grooves orchannels 704, 706 seen more clearly in the cross-sectional view. In theillustrated embodiment, pre-master mold 700 is fabricated using atwolevel photolithography technique similar to that described in FIG. 8.Topological features 706 have a greater depth than topological features704 and essentially traverse the entire thickness of photoresist layer708. In the illustrated embodiment, topological features 706 correspondto and form topological features in the replica molded elastomeric moldmaster which are alignment tracks, whose function is explained in moredetail below. Topological features 704 correspond to and formtopological features in the replica molded mold master which areresponsible for forming channels ultimately disposed in the second,upper level of the replica molded three-level membrane layer. It shouldbe understood that in alternative embodiments, one or more oftopological features 704 can comprise two-level topological featureshaving a first portion with a first depth with respect to surface 702and a second portion with a second, greater depth (e.g. corresponding tothe depth of topological features 706) with respect to surface 702. Forsuch embodiments, a replica molded top mold master would includetwo-level topological features in positive relief for forming channelsdisposed in the second, upper level of the replica molded membrane aswell as connecting channels traversing the membrane. In suchembodiments, the lower mold master can include channel-formingtopological features having a single, uniform height or can includechannel-forming topological features that are also two-level topologicalfeatures.

[0142] In Step 2, pre-master mold 700 is placed into the bottom ofcontainer 712. The container is then filled with a hardenable liquid,such as PDMS prepolymer, to a level at least covering upper surface 702of pre-master mold 700. Subsequently, the hardenable liquid is cured orsolidified, as previously discussed, and, in Step 3, is removed from thepre-master mold, optionally trimmed, and treated with a release agent,for example by silanization or oxidation followed by silanization. Theresulting structure 720 comprises a replica molded mold master includinga surface 722 having disposed thereon topological features 724 at afirst height with respect to surface 722 and corresponding totopological features 704 of pre-master 700, and topological features 726having a second, greater height with respect to surface 722 andcorresponding to topological features 706 on pre-master 700. Topologicalfeatures 724 comprise channel-forming features and topological features726 comprise alignment tracks.

[0143]FIG. 9b illustrates steps for forming the replica moldedthree-level membrane layer with the upper mold master 720 producedaccording to the steps outlined in FIG. 9a above and a lower mold master620 produced according to the method outlined previously in FIG. 8. InStep 4, a quantity of hardenable liquid 310, for example PDMSprepolymer, is placed in contact with upper surface 622 of lower moldmaster 620 in an amount sufficient to form a layer having a thickness atleast equal to the height of topological features 628 and 630. Uppermold master 720 is then brought into contact with lower mold master 620in Step 5 and is manually manipulated until topological features 726comprising alignment tracks in the upper mold master mate andinterdigitate with topological features 630 comprising alignment tracksin the lower mold master. Upon mating and interdigitating of alignmenttracks 726 and 630, the alignment and relative position ofchannel-forming topological features 724 of the upper mold master andchannel-forming topological features 624 of the lower mold master issuch that they are properly positioned and aligned with respect to eachother to form the desired three-dimensional microfluidic network channelstructure within the replica molded membrane layer. The interfacebetween the upper mold master 720 and lower mold master 620 during thereplica molding process in Step 5 is seen more clearly in thecross-sectional view. The cross-sectional view illustrates that, uponproper alignment, alignment tracks 726 of upper mold master 720 mate andinterdigitate with alignment tracks 630 in lower mold master 620. Inaddition, the cross-sectional view also clearly illustrates theconformal, sealing contact made between channel-forming feature 725 inupper mold master 720 and the upper surface of second portions 628 ofthe topological features on the surface of the lower mold master.

[0144] In Step 6, hardenable liquid 310, for example PDMS prepolymer, iscured, as previously described and upper mold master 720 is peeled awayfrom lower mold master 620. In the illustrated embodiment, where uppermold master 720 comprises silanized PDMS, lower mold master 620 has anupper surface 622 comprising polymeric photoresist and hardenable liquid310 comprises PDMS prepolymer, the replica molded PDMS membrane layer730 formed upon curing will adhere more strongly to surface 722 of uppermold master 720 than to surface 622 of lower mold master 620 and, uponpeeling away of upper mold master 720, will remain adhered to andsupported by upper mold master 720, thus preventing damage to themembrane.

[0145] Replica molded membrane layer 730 includes therein channels 732disposed within lower surface 734 of membrane 730, formed by firstportion 626 of topological features 624 of lower mold master 620; upperchannels 736 disposed within upper surface 738 of the membrane, formedby topological features 724 of the upper mold master; and connectingchannels 740 traversing the membrane and interconnecting surface 734 andsurface 738, which interconnecting channels are formed by secondportions 628 of two-level topological features 624 of lower mold master620. Thus, in the presently described method, a single replica moldedlayer is formed that includes therein all three levels required to forma three-dimensional microfluidic network structures according to theinvention. In addition, because of the provision of alignment tracks 726and 630, the entire three-dimensional network structure was formedwithout the need for performing an alignment of features or channelsrequiring the use of a microscope or micromanipulator. Because thepresent method does not require visual alignment of features orchannels, it can be especially useful for forming microfluidic membranestructures from materials that are opaque to visible light.

[0146] When, as illustrated, the three-level membrane is formed byutilizing one mold master formed via a photolithographic ormicromachining technique (e.g. mold master 620) together with anelastomeric mold master (e.g. 720), which is formed by replica molding apre-master mold formed via a photolithographic or micromachiningtechnique (e.g. pre-master 700), if the hardenable liquid utilized toform the replica molded mold master (e.g. as illustrated in Step 2 ofFIG. 9a) has a tendency to shrink during hardening, this shrinkageshould be taken into account when sizing and positioning the topologicalfeatures of the pre-master, so that topological features of the replicamolded mold master will properly match those of the other mold master toyield the desired alignment of channels. For example, when PDMS is usedto form one mold master, it has been found that the size and relativespacing of the features in the pre-master should be increased by about0.66% over that desired for the final PDMS mold master in order toaccount for shrinkage of the mold master during curing of thepre-polymer.

[0147] Replica molded polymeric membrane 730 can be removed from uppermold master 720 and can be utilized as a stand-alone structure or can bestacked with other such structures to form more complex networks.Optionally, and as shown in Step 7, before removal from upper moldmaster 720, lower surface 734 of membrane 730 can be brought intoconformal contact with a lower substrate layer 750, for example, a flatpiece of PDMS, silicon wafer, microchip, or other substrate, and canoptionally be sealed thereto as previously described. Substrate layers,instead of having flat smooth surfaces as illustrated, can, in otherembodiments, include topological features thereon that are matable withtopological features on the surface of the replica molded membrane, forexample, alignment tracks 739, so that, upon interdigitation of thematable topological features on the substrate layer and one or moretopological features on the surface of the replica molded membrane, themembrane is aligned and oriented in a desired configuration with respectto the substrate.

[0148] After contacting the membrane with the substrate layer and,optionally, essentially irreversibly sealing the membrane to thesubstrate layer, upper mold master 720 can then be removed from uppersurface 738 of membrane 730 as illustrated in step 8. The resultingmicrofluidic network structure 760 can be utilized as shown or aftertrimming away the regions of the membrane including alignment tracks739. Structure 760 is useful, for example, as a microfluidic membranestamp for patterning a material surface, the stamping surface comprisingupper surface 738 of membrane 730, which has channels 736 disposedtherein. Structure 760 is also useful for embodiments wherein themicrofluidic network structure is utilized as a mold in which to formthree-dimensional networks of materials having a structure correspondingto the channel structure in membrane 730, as described in more detailbelow.

[0149] For embodiments where it is desired to provide an enclosed seriesof microfluidic channels, upper surface 738 of membrane 730 issubsequently placed in conformal contact with and, optionally sealed to,an upper substrate layer 770. Upper substrate layer 770 can comprise aslab of PDMS or other substrate layer desirable for a particularapplication, as previously discussed. Also, as previously discussed,inlet and outlet conduits can be formed within either or both ofsubstrate layers 770 and 750 in order to interconnect the fluid flowpaths of the microfluidic channel structure to the external environment.

[0150]FIG. 9c illustrates a modification of the embodiment forfabricating the three-dimensional microfluidic structure, as illustratedin FIGS. 9a and 9 b. In the modification illustrated in FIG. 9C, theupper and lower mold masters utilized for forming the three-levelreplica molded membrane layer each include two-level topologicalfeatures thereon for forming the connecting channels traversing thereplica-molded membrane.

[0151] The two-level features of the upper and lower mold masters thatform the connecting channels through the membrane are configured to havecomplementary, mateable shapes, such that when the mold masters areplaced together during the replica molding step (e.g., step 5 asillustrated in FIG. 9b), the mateable, channel-forming topologicalfeatures on the upper and lower mold masters will mate/interdigitatewith each other, for example, as shown in FIG. 9c(iv). Providing suchmateable, connecting channel-forming features can reduce any tendency ofthe hardenable liquid for forming the replica molded membrane to beincompletely excluded from the regions forming the connecting channelsduring the molding process, which incomplete exclusion can lead to theformation of an undesirable, thin layer of hardened polymer occludingthe connecting channels after molding. In the modified embodimentillustrated, wherein the two-level topological features of the upper andlower mold master that form the connecting channels are configured withshapes that are mateable with each other, the hardenable liquid can bemore effectively and thoroughly excluded from the region molding theconnecting channels, thus effectively eliminating any tendency to form athin film of hardened material occluding the connecting channels uponformation of the membrane.

[0152] In addition, the mateable, connecting channel-forming two-leveltopological features can also serve a purpose similar to that of thealignment tracks discussed above. Namely, upon mating or interdigitationof the mateable, connecting channel-forming features of the upper andlower mold master, the alignment and relative position of the variousother channel-forming topological features of the upper and lower moldmaster will be properly positioned and aligned with respect to eachother to form the desired three-dimensional microfluidic network channelstructure within the replica molded membrane layer. Also, relativemotion between the upper and lower mold masters, leading tomisalignment, during the replica molding step can be reduced oreliminated. Accordingly, although the alignment track-forming featuresare illustrated in the modified embodiment shown in FIG. 9c, because themateably-shaped connecting channel-forming topological features of theupper and lower mold master can perform essentially the same functionand fulfil essentially the same purpose, in some embodiments utilizingthe modified mold masters, the alignment track-forming features could beeliminated.

[0153]FIG. 9c(i) and (ii) illustrate a modified pre-master mold 781 forforming the replica molded upper mold master 782 that includes thetwo-level connecting channel-forming topological features configured tomate/interdigitate with complementary connecting channel-formingfeatures in the lower mold master. Pre-master mold 781 can be fabricatedby forming topological features on surface 702, for example aspreviously illustrated in the context of FIGS. 8 and 9a. As previouslydescribed in the context of FIG. 9a, since it is desired that thetopological features formed in the replica molded top mold mastercomprised positive, high-level relief features protruding from thesurface of the mold master, the topological features formed on surface702 comprise negative, low-level relief features characterized bygrooves or channels 704, 706, and 783. In the illustrated embodiment,pre-master mold 781 is fabricated using a two-level formingphotolithography technique similar to that described above in FIG. 8.

[0154] One-level channel-forming feature 704, and two-level alignmenttrack forming feature 706 are essentially identical to those previouslydescribed in the context of FIG. 9a. In contrast to the embodimentillustrated previously in FIG. 9a, however, pre-master mold 781 includesa topological feature 784 corresponding to and forming a topologicalfeature in the replica molded mold master, which is responsible forforming a channel ultimately disposed in the second, upper-level of thereplica molded three-level membrane layer, which feature 784 includes,and is bounded by, topological features 783, which are configured toform connecting channel-forming features in the replica molded uppermold master that will have a shape that is mateable to complementaryconnecting channel-forming features in the lower mold master.Topological feature 783, shown in cross-section, comprises an outer ring785 in two-level negative relief surrounding a central post 786, thering and post together forming a “donut”-shaped two-level annulus.

[0155]FIG. 9c(ii) illustrates the resulting upper mold master formed byreplica molding pre-master 781, as discussed previously in the contextof FIG. 9a, Step 2. The resulting structure 782 comprises a replicamolded mold master including a surface 722 having disposed thereontopological features 724 and 787 at a first height 791 with respect tosurface 722 corresponding to topological features 704 and 784,respectively, of pre-master 781, and topological features 726 having asecond, greater height 794 with respect to surface 722 and correspondingto topological features 706 on pre-master 781. Upper mold master 782also includes topological features 788, corresponding to topologicalfeatures 783 of pre-master 781, features 788 including a central holeregion 790, in which the molded material comprising the mold masterextends to a position at the first height 791 with respect to surface722, and an outer peripheral ring 789 having a second, greater height794 with respect to surface 722. Topological features 788 comprisetwo-level, connecting channel-forming features, having a shape that ismateable to corresponding features on the lower mold master.

[0156] The lower mold master 792, illustrated in FIG. 9c(iii) issubstantially similar to lower mold master 620 illustrated and discussedpreviously in the context of FIGS. 8 and 9b, however, the secondportions (e.g., portions 628 as illustrated in FIG. 9b) of two-leveltopological features 626 of FIG. 9b, which are now called out by figurelabel 793, are somewhat smaller in diameter than those illustrated inFIG. 9b, and are sized and positioned to mate and interdigitate withholes 790 of interconnecting channel-forming topological features 788 ofupper mold master 782, when the mold masters are brought together andalligned for forming the three-level microfluidic membrane asillustrated in FIG. 9c(iv).

[0157] It should be understood that while, in the illustratedembodiment, the shape of the matable connecting channel-formingtopological features of the upper mold master comprises a circular,donut-shape annulus, and that of the lower mold master connectingchannel-forming topological features comprises a post, in otherembodiments, this configuration could be reversed such that theannulus-shaped features are present on the lower mold master and theposts are present on the upper mold master. In addition, in otherembodiments, upper mold master 782, as discussed previously, need not bea replica molded elastomeric structure, but instead could comprise amold master formed in photoresist, or other material, for examplesimilar to lower mold master 792, which could be formed by, for examplea micro-machining technique or, more preferably, as previously discussedin the context of FIG. 8.

[0158] It should also be understood that while the mateable,interconnecting channel-forming features illustrated in the presentembodiment comprise a circular cylindrical post-annulus arrangement, inother embodiments, the interdigitating, mateable shapes of theinterconnecting channel-forming features of the upper and lower moldmasters could be selected from an extremely wide variety of suitablymateable shapes. For example, instead of a circular post mating with anannulus having a circular centrally-disposed bore therein, a variety ofalternative cylindrical shapes could instead be utilized, for examplesquares, triangles, rectangles, n-sided polygons, ovals, etc.Alternatively, mateable configurations other than a post-annulusconfiguration, as illustrated, could be employed. For example, one ofthe mold masters could include interconnecting channel-forming featuresincluding a slot element that is mateable with a corresponding grooveelement in the interconnecting channel-forming features of the othermold master, or, alternatively, one mold master could provideinterconnecting channel-forming features including a halfcylinder-shaped element with the other mold master also providinginterconnecting channel-forming features including half cylinder-shapedelements, which half cylinders-shaped elements of the first and secondmold masters to mate together to together form cylindricalinterconnecting channel-forming features. Those of ordinary skill in theart will readily envision a wide variety of such mateable shapes andconfigurations suitable for use in the present context and providingsubstantially equivalent function and performance as described above.Each of such alternative configurations is deemed to be an equivalentstructure falling within the scope of the present invention.

[0159]FIG. 10 illustrates a method for forming the five-levelmicrofluidic network structure, shown previously in FIG. 4a, comprisinga coiled network of interconnected channels forming a first fluid flowpath surrounding a straight channel forming a second fluid flow path.The method in FIG. 10 is based upon the methods previously described inFIGS. 8, 9a, and 9 b discussed above. In the method shown in FIG. 10,two separate molded replica membrane layers are formed, which aresubsequently aligned with respect to each other and sealed together toform the final, overall, five-level coiled network structure 220. Thefirst molded replica membrane layer 800 comprises three levels of theoverall structure and a second molded replica membrane layer 810comprises the remaining two levels of the overall microfluidic networkstructure. Molded replica layer 800 comprising three levels is formed bythe membrane sandwich method previously discussed in the context of FIG.9b and utilizing a lower mold master 802 having formed thereon aplurality of two-level topological features 804 having first portions806, forming channels 807 disposed within the first, lowermost level ofthe overall microfluidic network structure, and second portions 808forming connecting channels traversing the level adjacent to andpositioned immediately above the lowermost level of the microfluidicnetwork structure upon replica molding.

[0160] Upper mold master 812 is preferably a replica molded elastomericmaterial (e.g. like mold master 720) and includes a bottom surface 814having a plurality of single-level topological features 816 protrudingtherefrom including a centrally disposed feature 818, forming thestraight channel 819 disposed on the second, upper level of membranelayer 800, and a plurality of features 820, aligned with second portions808 of topological features 804 of lower mold master 802, forming acontinuation of connecting channels 821 through the second, upper levelof replica molded layer 800 upon replica molding. Molded replica layer810, comprising the two-uppermost levels of the overall structure, isformed by the same membrane sandwich method utilizing lower mold master802 and an upper mold master 830, which comprises a flat slab ofpreferably elastomeric material. Two-level topological features 804,having first portions 806 and second portions 808, form a series ofchannels 832 disposed within lower surface 834 of molded replica layer810 and form connecting channels 833 traversing the thickness of moldedreplica layer 810, upon replica molding of layer 810.

[0161] In order to complete the assembly and form the overall coiledmicrofluidic network structure 220, molded replica layer 810 is rotated1800 in the direction of arrow 836, stacked on top of molded replicalayer 800, aligned so that the replica molded channels are registered toform the desired coiled channel network structure, brought intoconformal contact with, and optionally sealed to molded replica layer800. Optionally, surface 834 of molded replica layer 810 and/or surface809 of molded replica layer 800 can be brought into conformal contactwith, and optionally sealed to, a substrate layer (e.g., 838, 839) priorto or subsequent to stacking, aligning, and, optionally, sealing layers800 and 810 to each other. If desired, excess material comprising layers800 and 810 can be trimmed from the structure as illustrated in thefinal step of FIG. 10. The resulting structure 220 includes the coiled,two fluid flow path microfluidic network previously described in detailin the context of FIG. 4a above.

[0162] In addition to being useful as fluid flow directing networks forapplications requiring fluid management in very small scale devices, forexample, in micro total analysis systems (μTAS), the microfluidicnetwork structures provided according to the invention are also usefulfor a variety of other uses. For example, microfluidic channel systemsfabricated according to the invention can be used to fabricate a varietyof microstructures having three-dimensional structures corresponding toa three-dimensional network of channels within a microfluidic networkstructure. Such microstructures can be formed by filling the channelnetwork of the microfluidic systems with a hardenable liquid,solidifying the hardenable liquid within the network channels, and,optionally, removing the surrounding microfluidic network structure toyield a free-standing microstructure comprised of the solidifiedhardenable liquid. The hardenable liquid utilized for formmicrostructures that are replica molded within the inventivemicrofluidic network systems can comprise essentially any of thehardenable liquids described above as being useful for forming themicrofluidic network structures themselves. The hardenable liquidschosen to form the replica molded microstructures should be chemicallycompatible with the microfluidic network structure and, for embodimentswhere it is desired to selectively remove a surrounding microfluidicnetwork structure, should be resistant, once hardened, to whatevertreatment is required to dissolve or otherwise remove the surroundingmicrofluidic network structure. In one particular illustrative example,a microfluidic network structure produced according to the invention andcomposed of PDMS can be filled with an epoxy prepolymer, so that theepoxy prepolymer essentially completely fills the microfluidic channelstructure of the microfluidic network. The epoxy prepolymer can then becured, for example by exposure to ultraviolet light through thesurrounding PDMS microfluidic channel structure, in order to cure theepoxy prepolymer and form a solid microstructure within the channels.The surrounding PDMS microfluidic network can then be dissolved, forexample with tetrabutylammonium fluoride (1.0 M in tetrahydrofuran)leaving behind a free-standing microstructure, comprised of epoxypolymer, having a three-dimensional structure corresponding to thethree-dimensional network of channels in the PDMS microfluidic channelstructure.

[0163] In another illustrative application for certain microfluidicchannel structures provided by the invention, the microfluidic channelstructure is used as a three-dimensional microfluidic applicator or“stamp” for forming a pattern on a material surface corresponding to apattern of channels disposed in one level of the microfluidic networkstructure. The “stamping surface” of such structures includes disposedtherein a series of channels forming indentations, which channels candeliver material to a substrate surface in contact with the “stampingsurface” in order to form a pattern thereon corresponding to the patternof channels in the stamping surface. Examples of structures discussedpreviously having “stamping surfaces” are microfluidic channel structure560 illustrated in FIG. 7 having a stamping surface 554, andmicrofluidic channel structure 760 illustrated in FIG. 9b having astamping surface 738.

[0164] The method for patterning a material surface with a microfluidicnetwork structure provided according to the invention comprisescontacting a stamping surface of the microfluidic network structure witha material surface to be stamped, and, while maintaining the stampingsurface in contact with the material surface being stamped, at leastpartially filing one or more flow paths of the microfluidic channelstructure with a fluid so that at least a portion of the fluid contactsthe material surface. Subsequently, if desired, the stamping surface canbe removed from the material surface, yielding a pattern on the materialsurface, according to the pattern of channels disposed within thestamping surface, formed by contact of the material surface with thefluid.

[0165] One example of such a stamped pattern is illustrated in FIG. 11.The microfluidic stamp utilized for forming the pattern in FIG. 11 waspreviously illustrated in FIG. 1a. In forming the pattern in FIG. 11,microfluidic network 100 (FIG. 1a) is formed so that lower surface 134is configured as a stamping surface, with the channels disposed thereincomprising indentations within the surface exposed to the externalenvironment. For embodiments wherein the microfluidic network structuresare utilized as stamps/applicators, it is especially preferred that themicrofluidic network structures be formed of an elastomeric material, sothat the stamping surface of the stamp is able to make a fluid-tightconformal seal with a wide variety of shapes and textures of materialsurfaces.

[0166] The microfluidic stamps provided according to the invention canbe utilized to form patterns on material surfaces comprised of anextremely wide variety of materials, as would be apparent to those ofordinary skill in the art. The structures provided according to theinvention, when used as stamps, can be utilized, for example: to formpatterned self-assembled monolayers (SAMs) on material surfaces; to formpatterns of inorganic materials on surfaces; to form patterns of organicand/or biological materials on surfaces; to form patterns on surfacesvia contacting the surfaces with a material that chemically reacts withand/or degrades/etches the material surface; etc. Essentially anymaterial able to be printed via conventional microcontact printingtechniques can be patterned onto a surface using the inventivemicrofluidic stamping structures provided by the invention. A variety ofsuch materials and applications is described in detail in U.S. Pat. Nos.5,512,131; 5,620,850; 5,776,748; 5,900,160; 5,951,881; and 5,976,826,each of which is incorporated herein by reference.

[0167] The microfluidic stamping structures provided according to theinvention have several advantages over traditional two-dimensionalmicrofluidic stamps. For example, the microfluidic stamping structuresprovided according to the invention have the ability to simultaneouslyform a plurality of patterns onto a material surface, each of whichpatterns is comprised of a different material or “ink”. In general, thenumber of different patterns and materials which can be patterned onto amaterial surface simultaneously by the stamps provided according to theinvention is equal to the number of independent, non-fluidicallyinterconnected fluid flow paths disposed within the microfluidicstamping structure.

[0168] In order to form multiple patterns with different “inks”utilizing traditional two-dimensional microcontact printing stamps,individual stamps each having a separate pattern thereon must beutilized, with each stamp being inked with a different fluid, and witheach pattern being carefully overlaid upon the previous pattern andaligned thereto. By utilizing the three-dimensional microfluidic channelstructures provided according to the invention, the inventive stamps areable to form, simultaneously, essentially any desired number ofarbitrarily complex patterns on a material surface using a single stampin a single stamping step.

[0169] For example, referring again to FIG. 11, the microfluidic channelsystem of FIG. 1a having a stamping surface 134 is able tosimultaneously form an overall pattern on material surface 900corresponding to seven discrete subpatterns (A-G), each subpatterncorresponding to channels disposed within stamping surface 134 of one ofthe seven fluid flow paths (102, 104, 106, 108, 110, 112, 114) of themicrofluidic channel system shown in FIG. 1a. As illustrated, each ofsubpatterns A-G includes discrete pattern features (902, 904, 906, 908,910, 912, 914, 916, 918, 920, 922, and 924) which are non-continuous,and which are non-intersecting with each other. In general, themicrofluidic stamps provided according to the invention are capable offorming patterns comprised of discrete regions, wherein the discreteregions are non-continuous with each other, and wherein discrete regionscorresponding to and formed by channels within the stamping surface ofthe structure corresponding to two different non-fluidicallyinterconnected fluid flow paths are non-intersecting with each other.

[0170] In the illustrated pattern shown in FIG. 11, it is possible topattern up to seven different materials (“inks”) onto material surface900 simultaneously using microfluidic stamp 100 by filling each of theseparate flow paths of the microfluidic network with a different fluidafter contacting stamping surface 134 with material surface 900. Forexample, patterned regions labeled “A” in FIG. 11 can comprise a firstpatterned material, regions labeled “B” can comprise a second patternedmaterial, regions labeled “C” can comprise a third patterned material,regions labeled “D” can comprise a fourth patterned material, regionslabeled “E” can comprise a fifth patterned material, regions labeled “F”can comprise a sixth patterned material, and regions labeled “G” cancomprise a seventh patterned material. The overall pattern that resultson material surface 900 corresponds to each of the seven individualsubpatterns (A-G) formed by contact of material surface 900 with theparticular fluids contained within each of the individual flow pathsforming subpatterns A-G.

[0171] In some embodiments, regions of stamping surfaces disposedbetween channel indentations that make conformal contact with thematerial surface being stamped can also, if desired, be coated withanother material or, “ink”. In such embodiments, in addition to formingpatterns corresponding to the channel structures in the stamping surfaceas described above, the regions surrounding, contiguous with, andseparating the patterns formed by the channel structures (“printingregions”) can also contain a deposited material, carried by the printingregions, which material is printed on the material surface uponconformal contact of the “printing regions” of the stamping surface withthe material surface. The above technique enables an operator toessentially simultaneously perform a conventional microcontact printingstep and a step of depositing material in a predetermined pattern on thematerial surface via the channels disposed in the stamping surface ofthe microfluidic stamp.

[0172] Because it is possible to create arbitrarily complex patternscomprising a large number of patterned regions containing differentpatterned materials, the stamps provided according to the inventionpotentially have an extremely wide range of use for a wide variety ofapplications. For example, in one preferred application, the inventivestamps can be utilized to pattern cells and/or proteins onto surfaces.For example, proteins can be selectively patterned onto a surface whichare adhesive to cells, non-adhesive to cells, or selectively adhesive tocertain cells while non-adhesive to other cells. By forming patternswith such proteins, complex patterns of one cell type or a variety ofcell types can be selectively patterned onto surfaces for variousapplications, for example, for forming biosensors or performing drugscreening tests. With the microfluidic stamps provided according to theinvention it is possible, in principle, to pattern a large number, forexample in excess of 200 or 300, different cell types, each separatedfrom each other and arranged in a patterned array format. Suchpatterning can be accomplished, according to the invention, by, forexample, selectively patterning proteins onto a surface adherent toparticular cell types followed by contact of the patterned materialsurface with one or more cell suspensions, or by selectively patterninga plurality of different cell types onto a surface directly using amicrofluidic stamp and filling particular fluid flow paths within thestamp with suspensions containing a discrete cell type or mixture ofcell types desired to be patterned onto the surface. The ability to formpatterns comprising arrays of regions, with each region including aparticular cell type or mixture of cell types, can enable the creationof material surfaces for use in biosensors or drug screening deviceshaving cells patterned thereon that can be easily and readily identifiedby their spatial locations on the surface.

[0173] Proteins can also be deposited, using the inventive microfluidicstamps, that tend to prevent or inhibit cell adhesion to a materialsurface. Such proteins are well known to those of ordinary skill in theart and include for example bovine serum albumin (BSA). In addition,proteins can be patterned according to the invention that tend topromote cell adhesion to the material surface. Such proteins include,for example, fibrinogen, collagen, laminin, integrins, antibodies,antigens, cell receptor proteins, cell receptor antagonists, andmixtures of the above.

[0174] As described above, the microfluidic stamping structures providedaccording to the invention, can be utilized to deposit a patterned layerof cells on a material surface. Cells which can be patterned on materialsurface comprise essentially the entire range of biological cellsincluding, but not limited to, bacterial cells, algae, ameba, fungalcells, cells from multi-cellular plants, and cells from multi-cellularanimals. In some preferred embodiments, the cells comprise animal cells,and in some such embodiments comprise mammalian cells, such as humancells.

[0175] In one preferred embodiment, the mammalian cells compriseanchorage dependent cells, which can attach and spread onto materialsurfaces. In one preferred embodiment, the microfluidic network stampingstamp provided according to the invention is placed with its stampingsurface in conformal contact with the material surface to be patternedwith a plurality of cells, and, after filling one or more fluid flowpaths of the microfluidic stamp with one or more suspensions of cellsand before removing the stamp from the material surface, the cells areallowed to incubate within the channel structure of the microfluidicstamp for a period of time sufficient to allow the cells to attach andspread onto the material surface. In such an embodiment, the shape orpattern of channels can be specifically designed to have a predeterminedarchitecture or pattern selected to simulate a desired tissuemicro-architecture in order to study the relationship between cell shapeand/or position and cell function.

[0176] In other embodiments, two or more different cell types can bepatterned onto a material surface, as described above, and, subsequentto removing the microfluidic stamp, can be allowed to grow upon thesurface and spread such that cells of the two or more different cellstypes spread together and come into contact on the surface after aperiod of time has elapsed. Such a patterning and incubation method canbe useful as part of an in vitro assay, which is able to determineand/or study interactions between different cell types. For example,such method can form part of an in vitro assay able to determine anangiogenic potential of a particular type of tumor cell. In oneparticular application contemplated, two different cell types comprisingcapillary endothelial cells and tumor cells are patterned onto amaterial surface and allowed to grow and spread upon the surface afterpatterning, as described above, in order to simulate and studyangiogenesis during tumor formation. In vivo, tumor cells tend toattract and direct the growth of capillary endothelial cells to form newblood vessels to supply nutrients and oxygen for tumor growth. Byforming a defined pattern of capillary endothelial cells and tumor cellsutilizing the microfluidic stamps provided according to the invention,it can be possible to enable assays able to study the differential andcompetitive attraction of capillary endothelial cells to different tumorcell lines. This technique, enabled by the present invention, can leadto the development of a simple, standardized, and quantitative in vitroassay for comparing the angiogenic potential of different tumor cells.

[0177] In addition, as discussed above, the present microfluidic networkstamps enable two or more different cell types to be patterned onto amaterial surface in a wide variety of patterns of arbitrary complexityand in a predetermined arrangement, which arrangement can be selected tosimulate a distinct micro-architecture defined by the topologicalrelationship between the different cell types patterned on the surface.The ability to pattern and selectively deposit different cell types inwell-defined patterned structures, enabled by the present invention, canenable assays designed to study the functional significance of tissuearchitecture at the resolution of individual cells, and can enableassays designed to study the molecular interactions between differentcell types that underlie processes such as embryonic morphogenesis,formation of the blood-brain barrier, and tumor angiogenesis.

[0178] The function and advantage of these and other embodiments of thepresent invention will be more fully understood from the examples below.The following examples are intended to illustrate the benefits of thepresent invention, but do not exemplify the full scope of the invention.

EXAMPLE 1 Fabrication of a Mold Master by Multi-Level Photolithography

[0179] A mold master of photoresist on silicon having two levels offeatures in positive, high relief (i.e., protruding above the surface ofthe silicon wafer) was fabricated using the two-level photolithographytechnique outlined in FIG. 8. Designs for the channel systems for thefirst and second levels were generated with a CAD computer program(Free-Hand 8.0, MacroMedia, San Francisco, Calif.). High resolution(3386 dpi) transparencies were made by printing with a commercialprinter (Linotype, Hercules Computer Technology, Inc., Freemont, Calif.)from the CAD computer files. Two transparencies were produced, the firstcomprising the photomask for producing-features in the first level ofthe mold master and the second comprising photomask for producing thefeatures in the second level of the mold master.

[0180] Negative photoresist (SU8-50, Microlithography Chemical Corp.,Newton, Mass.) was spin-coated (at about 5,000 rpm for 20 sec) on asilicon wafer to a depth of about 50 μm and soft-baked at about 105° C.for about 5 min to drive off solvent from the spin-cast photoresist. Thefirst transparency was then used as a photomask and the photoresist wasexposed to UV radiation for about 45 sec (wavelength of spectral linesabout: 365 nanometers, 406 nanometers, and 436 nanometers at anintensity of about 10 mW/cm²).

[0181] Without developing the uncrosslinked photoresist, a second layerof photoresist was spin-cast to a depth of about 100 μm on top of thefirst layer. The second transparency comprising the second photomask wasaligned to the exposed features of the photoresist of the first layerusing a Karl Suss mask aligner and exposed to the UV radiation for about1 min. The silicon wafer containing the exposed photoresist layers wasthen hard-baked for about 5 min. at about 105° C. The second photomaskcontained the pattern corresponding to the interconnecting channels thatwould eventually link channels of the first, lower level formed by thefeatures exposed through the first photomask, and channels of the upperlevels of the replica molded structure ultimately molded with the moldmaster. As illustrated in FIG. 8, each of the photomasks also included apattern for forming alignment tracks surrounding the channel system.

[0182] Both layers of photoresist were developed at the same time toremove uncrosslinked photoresist with propylene glycol methyl etheracetate. The resulting bottom master included tall alignment featuresand channel features comprising two-level topological features inpositive relief. The surface of the bottom mold master including thetopological features was then silanized by placing the mold master in avacuum chamber with a few drops oftridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United ChemicalTechnologies, Inc., Bristol, Pa.) for about 2 hours. Silanization of themaster facilitates the removal of a PDMS replica after molding.

EXAMPLE 2 Fabrication of a Three-Dimensional Microfluidic NetworkIncluding a System of Channels in a “Basketweave” Configuration

[0183] In the following example, the method outlined in FIGS. 9a and 9 bwas utilized to produce a microfluidic network structure including achannel pattern therein having a basketweave structure similar to thatillustrated in FIG. 1a. First, a bottom master was produced as describedabove in Example 1 having disposed thereon two-level topologicalfeatures for forming channels within the molded replica arrangedsimilarly to those shown schematically in FIG. 12a by bottom master1000. The second step of the process comprised formation of a top masterincluding features for forming channels in the uppermost level of thereplica molded membrane. A similar schematic arrangement of features forproducing the channels, and the way in which the channels of the uppermold master and lower mold master fit together to mold the overallstructure, is also illustrated in FIG. 12a, making specific reference toupper mold master schematic 1002.

[0184] The top mold master was made by first fabricating a two-levelstructure in photoresist on silicon comprising a pre-master by a methodsimilar to that discussed above in Example 1. The pre-master containedfeatures in negative, low-relief (i.e., comprising indentations belowthe level of the bulk surface) so that replica molding the upper moldmaster with the pre-master produced features in positive, high-relief onthe upper mold master, as shown schematically in FIG. 12a and as shownand discussed earlier in the context of FIG. 9a. The topologicalfeatures of the pre-master corresponding to the channel system extendedto a level below the surface of the photoresist, but did not traverse itcompletely; these features were all on one level. Alignment tracks (notshown in FIG. 12a) that were shaped and positioned to form alignmenttracks in the replica molded top mold master that fit between alignmenttracks on the bottom master (not shown in FIG. 12a) during replicamolding of the microfluidic membrane with the mold masters werefabricated in deeper, negative relief and went all the way through thephotoresist to the silicon wafer. The pre-master was then silanized asdescribed above in Example 1. The pre-master was then covered with PDMSprepolymer (Sylgard 184™ silicone elastomer with about a 1:10 ratio ofcuring agent to elastomeric silicone polymer) and cured at about 75° C.for about 1 hour. The PDMS replica, comprising a top mold master, wasthen peeled from the pre-master, trimmed, and oxidized in a plasmacleaner (PDC-23G, Harrick, Ossining, N.Y.) for 1 min, and then wassilanized by placing it in a vacuum chamber with a few drops oftridecafluoro-1,1,2,2-tetrahydrooctyl-1-trichlorosilane (United ChemicalTechnologies, Inc., Bristol, Pa.) for about 8 hours.

[0185] The upper mold master was then placed facedown on top of thesurface of the bottom mold master including topological features, with adrop of PDMS prepolymer in between. The features of the masers werealigned quickly and without magnification by manually sliding the topmaster over the prepolymer and bottom master until its tall alignmenttracks slipped between the tall alignment tracks of the bottom master.Utilizing PDMS for the top master enabled visual observation of thefeatures of the masters and made alignment straightforward. A microscopewas not necessary because the alignment tracks were macroscopic. Inaddition to facilitating the alignment of the segments of the channelsystem quickly and without magnification, the alignment tracks alsobalanced the top master and prevented the registered masters fromshifting in position in response to physical disturbances or theapplication of pressure during molding.

[0186] A pressure of about 100 g/mm² (1000 kPa) was then applied to thetop master so that prepolymer did not seep between features that were incontact, and the PDMS was heated to about 75° C. and cured in place forabout 1 hour. In addition, two flat pieces of PDMS comprising an upperand lower substrate layer were formed by casting the PDMS prepolymeragainst a flat, silanized silicon wafer and curing, as described above.To transfer the membrane, the membrane and top master were peeled off asa single unit from the bottom master; the surface of the membrane andthe flat pieces of PDMS were oxidized in an air plasma for 1 min, asdescribed above; and the oxidized surfaces were then brought togetherimmediately. The oxidized PDMS surface remains reactive for a fewminutes after plasma treatment. Reactivity of the surface can beprolonged by covering the surface, if desired, with a hydrophilic liquidsuch as water, methanol, trifluoroethanol, or mixture thereof. Aprotected surface will still seal more than 30 min after oxidation.

[0187] After contacting the membrane with the bottom PDMS slab, the topmaster was peeled off, and the top surface of the membrane was sealed tothe second oxidized flat slab to enclose the channel system. The entirestructure was then trimmed to a convenient size. The resulting structureincluded a microfluidic network incorporating eight channels in thex-direction and eight in the y-direction, each having a width of about100 μm and a height of about 70 μm, and each alternating betweencrossing over and under channels oriented perpendicular to themselves.The entire structure had a total area in the x-y plane of about 30 mm²and contained 64 crossovers.

[0188]FIG. 12b is a photocopy of an optical photomicrograph showing anen face phase contrast image of the structure as viewed in the negativez-axis direction. The optical micrograph illustrated in FIG. 12b wastaken of the replica molded membrane alone prior to sealing the membranebetween the upper and lower PDMS substrate layers. The opticalphotomicrograph clearly shows the basketweave microfluidic channelstructure and the crossover points of the channels, appearing asintersections in photographed the x-y plane.

[0189] After enclosing the membrane between an upper and lower PDMSsupport layer as described above, flow paths extending in the ydirection were filled with a solution of fluorescein and flow pathsextending in the x direction were filled with a solution of Meldola'sBlue Dye. FIG. 12c is a photocopy of a photomicrograph of themicrofluidic channel system filled as described above, with the observerviewing the system en face in the negative z-axis direction. FIG. 12cshows, without ambiguity, which channels cross over and which crossunder each other, and also demonstrates that the channels do notintersect, as would be evidenced by mixed colors at any point.

EXAMPLE 3 Fabrication of Microstructures by Replica Molding With aMicrofluidic Network Structure

[0190] A microfluidic membrane including a three-level channel system ina basketweave pattern was produced as described in Example 2. Themicrofluidic membrane was placed upon a flat PDMS slab so that the uppersurface of the PDMS slab and the lower surface of the membrane were inconformal contact but were not irreversibly sealed to each other. Theupper surface of the membrane was left open to the atmosphere. An epoxyprepolymer (EP-TEK, Epoxy Technology, Billerica, Mass.) was then placedat the channel openings and allowed to fill the channel structure bycapillary action. After approximately 1 hour standing at ambientpressure, the epoxy had degassed and filled the channels completely. Thefilled channels were then exposed to UV light (as described above inExample 1) for about 20 min through the PDMS. The surrounding PDMSmicrofluidic membrane was then dissolved in tetrabutylammonium fluoride(1.0 M in tetrahydrofuran). FIG. 12d is a photocopy of a scanningelectron photomicrograph of the resulting microstructure produced by thecured epoxy polymer.

EXAMPLE 4 Fabrication of a Microfluidic Network Structure Including aCoiled Fluid Flow Path Surrounding a Straight Channel

[0191] To demonstrate the capability of stacking, registering, andsealing membranes to each other to make structures having more thanthree levels of channels, a structure was fabricated including astraight channel surrounded by a coiled fluid flow path comprising aseries of interconnected channels. The flow path comprising the straightchannel was separated from the channels comprising the coiled flow pathby a thin, about 65-100 μm, PDMS layer. Examples of microfluidic systemsthat benefit from such a configuration include heat exchange elements orcountercurrent extraction system taking advantage of the diffusion ofsmall molecules across the PDMS layer separating the straight channeland the coiled fluid flow path. Multi-layer fabrication techniques suchas the one in the current example also have utility for devices forsorting and binding particles, and for complex channel network systemsthat have specific size constraints.

[0192] The method used for producing the five-level channel system bystacking and aligning two replica molded multi-level membranes wasillustrated above in FIG. 10. Referring to FIG. 10, first, bottom master802 was fabricated as described above in Example 1. Upper mold masters820 and 830 were fabricated as described in Example 2. Replica moldedmembranes 800 and 810 were fabricated of cured PDMS prepolymer, also asdescribed above in Example 2. Bottom master 802 was removed from each ofthe membranes and flat slabs of PDMS were sealed in their place, asdescribed above in Example 2. The top masters were then peeled off andthe two membranes were aligned face-to-face on the stages ofmicromanipulators. This orientation required that the two-level membrane810 be flipped over. The membranes were brought together and aligned,and were then backed apart by about 3 to about 5 mm without disturbingthe previous alignment. The separated membranes were then oxidized in anair plasma, as described above, and then brought into conformal contact.

[0193]FIG. 13 shows a photocopy of an optical photomicrograph of theresulting channel system as viewed en face along the negative z-axisdirection. Prior to the photomicrograph being taken, the two fluid flowpaths of the system were filled with a fluorescein solution, asdescribed in Example 2, to aid visualization of the channel system.

EXAMPLE 5 Fabrication of a Microfluidic Stamp and Etching of a Si/SiO₂Surface and Visualization of the Etched Surface Using OpticalInterference Colors

[0194] For the present example, a three-dimensional microfluidic stampwas produced according to the method outlined in FIG. 7. Referring toFIG. 7, two-level lower mold master 520 was prepared as previouslydescribed in Example 1 and one-level mold master 500 was prepared alsoas described in Example 1, except utilizing only a single layer ofphotoresist and a single photomask to produce only one level oftopological features. The top PDMS slab 510 was fabricated by placingmold master 500 in a container with surface 502 facing up, covering themold master with PDMS prepolymer, curing the PDMS prepolymer, asdescribed above in Example 2, and removing and trimming the moldedreplica to form PDMS slab 510.

[0195] PDMS membrane 550 was fabricated by sandwiching a drop of PDMSprepolymer between master 520 and a PTFE sheet. Pressure of betweenabout 10 and about 50 kPa was applied tending to force the PTFE sheetand mold master 520 together. The PDMS prepolymer was then cured, asdescribed in Example 2. After curing, PTFE sheet 540 was peeled away,leaving the membrane remaining attached to mold master 520 by van derWaals interactions.

[0196] To align and seal the PDMS slab to the PDMS membrane amicromanipulator stage was used. The slab and membrane were mounted onthe micromanipulator stage so that surface 514 was facing surface 556.The surfaces were brought into close proximity and aligned. Afteralignment, the surfaces were backed away from each other by a fewmillimeters using the micromanipulator. The entire alignment stage wasthen placed in a plasma cleaner (Anatech, Model SP 100 Plasma System,Springfield, Va.) and oxidized for about 40 sec in an oxygen plasma. Thepower level of the plasma cleaner was about 60 watts and the oxygenpressure was about 0.2 Torr. Sealing of the two layers was accomplishedby removing the assembly from the plasma cleaner and immediatelybringing the two aligned and oxidized PDMS surfaces into contact.

[0197]FIG. 14a illustrates schematically the channel system disposed inthe upper level 1010 of the microfluidic stamp and the lower level 1012of the microfluidic stamp, which lower level having a lower surface 554comprising the stamping surface. Surface 554 was brought into conformalcontact with material surface 1014 of substrate 1016. FIG. 14b is aschematic diagram illustrating the layout and interconnectivity of thethree-level channel system within microfluidic stamp 560 and theconfiguration of each of the three non-fluidically interconnected fluidflow paths 561, 563, and 565.

[0198] To create the etched pattern on surface 1014 shown in FIG. 14c,surface 554 of the microfluidic stamp was brought into conformal contactwith surface 1014 (comprising a Si/SiO₂ surface) and gentle pressure wasapplied to the stamp. Three aqueous solutions containing three differentconcentrations of hydrofluoric acid (10%, 5%, and 3% hydrofluoric acid,buffered at about pH 5 with a 6:1 ratio of NH₄F/HF) were allowed to flow(−1 cm/sec), with each solution confined to one of the non-fluidicallyinterconnected flow paths in the structure. Each of the channels in thestructure had a cross-sectional area, measured in a plane perpendicularto the channel's longitudinal axis, of about 500 μm². Where thehydrofluoric acid solutions came into contact with the surface, theyetched away the SiO₂. The rate of etching of SiO₂ for 10% hydrofluoricacid is about 20 nm/min. The lower concentrations etched at a rateproportionally less than the most concentrated solution. Thehydrofluoric acid solutions were flowed through the channels for aperiod of about 26 min before removing the stamp from the surface andvisualizing the pattern.

[0199] The optical interference color of an SiO₂ layer is very sensitiveto the thickness of the layer; a difference of about 30 nm, for example,can change the color from, for example, light green to blue. Thus,patterns etched to different depths within surface 1014 appear asdifferent colors. Referring to FIG. 14c, patterned features 1018,corresponding to fluid flow path 561, which contained the 10%hydrofluoric acid solution, were etched into surface 1014 to a depth ofabout 520 nm and appear green. Etched patterned features 1020,corresponding to fluid flow path 565, which contained the 5%hydrofluoric acid solution, were etched into surface 1014 to a depth ofabout 390 nm and appear yellow. Patterned features 1022, correspondingto fluid flow path 563, which contained the 3% hydrofluoric acidsolution, were etched into surface 1014 to a depth of about 70 nm andappear brown.

EXAMPLE 6 Patterned Deposition of Proteins Onto a Surface Using aThree-Dimensional Microfluidic Stamp

[0200] A microfluidic stamp having a stamping surface with spirallyarranged channels therein was produced by a method similar to thatdescribed above in Example 5. The microfluidic stamp had a microfluidicchannel system shown schematically in FIG. 15a. The stamp included twonon-fluidically interconnected fluid flow paths 1030 and 1032. Thechannels of fluid flow paths 1030 and 1032 are disposed in the stampingsurface of the microfluidic stamp in a nested spiral arrangement asillustrated in FIG. 15a.

[0201] The stamping surface of the microfluidic stamp was placed inconformal contact with a polystyrene surface of a petri dish. Spiralflow paths 1030 was then filled with a FITC-labeled bovine serum albumin(BSA) solution having a labeled BSA concentration of 1 mg/ml inphosphate buffer (pH 7.4). Fluid flow path 1032 was filled with aFITC-labeled fibrinogen solution containing 0.1 mg/ml labeled fibrinogenin phosphate buffer (pH 7.4). The proteins were allowed to absorb ontothe polystyrene surface for about 45 min. The channels were then flushedthoroughly with phosphate buffer; the stamp was peeled off; and thesurfaces were observed en face with fluorescence microscopy.

[0202]FIG. 15b is a photocopy of a photomicrograph taken of the surfaceof the petri dish as viewed utilizing fluorescence microscopy. Spiralpattern 1034 comprises a layer of deposited labeled BSA and spiralpattern 1036 comprises a layer of deposited labeled fibrinogen. Spiralpattern 1034 is brighter and more fluorescent because the concentrationof BSA used was about 10 times higher than the concentration offluorescently labeled fibrinogen.

EXAMPLE 7 Patterned Deposition of Cells Onto Surfaces Using TwoDifferent Microfluidic Systems

[0203] Cell cultures: Bovine adrenal capillary endothelial cells (BCEs)were cultured as described in J. Folkman, C. C. Haudenschild, B. R.Zetter, Proc. Natl. Acad. Sci. USA, Vol. 76, pp. 5217-5221, 1982. Inbrief, BCEs were grown in low glucose DMEM cell culture mediumsupplemented with 10% calf serum and 2 ng/ml basic fibroblast growthfactor (bFGF), and kept in a 10% CO₂ atmosphere. Human bladder cancercells (ECVs) from the ECV304 cell line were cultured in DMEMsupplemented with 10% fetal bovine serum (FBS) and kept in a 5% CO₂atmosphere. Cells from both cell types were labeled fluorescently beforeharvest at 37° C. in the CO₂ incubator. BCEs were incubated with1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI)-conjugatedacetylated low-density lipoprotein at 4 μg/ml, which is actively takenup by BCEs and stored in endosomal granula. ECV304 cells were incubatedwith 5 μM 5-chloromethylfluorescein diacetate (CMFDA), which reacts withintracellular glutathione. Before patterning, cells were washed withPBS, dissociated from the culture plates to which they were attachedduring culture with typsin/EDTA, washed with DMEM, and resuspended inthe respective culture media at a density of about 10⁶ cells/ml. Forculturing patterned cells (both BCEs and ECVs) after removal of the PDMSstamp, DMEM supplemented with 5% calf serum, 5% FBS, and 2 ng/ml bFGFwas used, and the cells were kept in a 10% C0 ₂ atmosphere.

[0204] Patterning: To form the first pattern of deposited cells, amicrofluidic stamp having the channel network structure illustratedschematically in FIG. 16a was fabricated by a method similar to thatdescribed above in Example 5. A stamping surface of the microfluidicstamp included disposed therein channels comprising a concentric squarepattern. The microfluidic stamp included three non-fluidicallyinterconnected fluid flow paths 1040, 1042, and 1044, fluid flow path1040 in fluid communication with outermost concentric square pattern1041, fluid flow path 1042 in fluid communication with the intermediateconcentric square pattern 1043, and fluid flow path 1044 in fluidcommunication with the innermost concentric square pattern 1045.

[0205] Before use, the PDMS microfluidic stamp was autoclaved at about121° C. for about 20 min, and the walls of the channels were coated withBSA by filling the channels with a 0 mg/ml BSA solution in pH 7.4phosphate buffer for about 1 hour before removing the solution andflushing with BSA-free phosphate buffer. The stamping surface was thenbrought into conformal contact with the surface of a polystyrene tissueculture dish. Suspensions of cells (at a concentration of about 5×10⁶cells/ml) were introduced into the three fluid flow paths and wereallowed to sediment and attach to the surface of the tissue culturedish. The cells used were BCEs and an ECV cell line (ECV-304). Beforebeing deposited, the BCEs were labeled with Dil-conjugated acetylatedlow-density lipoprotein, which was actively taken up by the BCEs andstored in their endosomal granula, and the ECVs with CMFDA, whichreacted with their intracellular glutathione. The BCE cell solutionswere introduced into fluid flow paths 1040 and 1044, and the ECV cellsolution was introduced into fluid flow path 1042. After introducing thecell suspension into the fluid flow paths of the microfluidic stamp, thecells were cultured for about 24 hours with the microfluidic stamp inplace on the tissue culture dish surface, so as to form a confluentlayer of cells on the surface of the tissue culture dish. After culture,the microfluidic stamp was removed from the surface, and the surface,having cells attached thereto, was immersed in tissue culture media, aspreviously described.

[0206]FIG. 16b is a photocopy of a photomicrograph of surface of thepetri dish as observed by fluorescence microscopy. The deposited BCEcells are attached to the surface in the outermost concentric squarepattern 1046 and the innermost concentric square pattern 1048. Suchcells, when viewed with the fluorescence microscope appear red in color.The ECV cells are deposited on the surface in concentric square pattern1050 and fluoresce green when viewed with the fluorescence microscope.FIGS. 16c and 16 d are photocopies of photomicrographs of the patternedsurface as viewed with phase-contrast microscopy, illustrating themorphology and arrangement of the cells within each of the patterns onthe surface.

[0207]FIGS. 17a and 17 b show the results of a similar cell patterningexperiment wherein two types of cells were deposited in achessboard-like pattern. The chessboard-like pattern was designed as ademonstration of the potential of the microfluidic stamping system andmethod of the invention to deposit multiple cell types in an arrayformat appropriate for a biosensor or drug screening applications. Insuch an array, the responding cells could be identified by their spatiallocation.

[0208] A microfluidic stamp having fluid flow paths shown schematicallyin FIG. 17a was prepared by a method similar to that described above inExample 5. The microfluidic stamp included eight non-fluidicallyinterconnected independent flow paths 1060, 1062, 1064, 1066, 1068,1070, 1072, and 1074. Each of the flow paths is in fluid communicationwith two square channels disposed in the stamping surface of themicrofluidic stamp. For example, fluid flow path 1060 is in fluidcommunication with square channels 1076 and 1078 disposed within thestamping surface of the microfluidic stamp.

[0209] A chessboard pattern of cells is shown in FIG. 17b, which is aphotocopy of a fluorescence photomicrograph. The patterned surface wasproduced using the same procedures used for patterning the concentricsquare pattern of FIGS. 16b-16 d. The two cell types used, BCEs andECVs, were fluorescently labeled, as described above, before beingdeposited onto the surface of a tissue culture plate. Solutions offluorescently labeled ECV cells were used to fill fluid flow paths 1060,1062, 1064, and 1066, and solutions of fluorescently labeled BCE cellswere used to fill fluid flow paths 1068, 1070, 1072, and 1074. The cellswere cultured with the stamp in place on the surface for 42 hours untila confluent layer of cells were formed on the surface of the tissueculture plate. The fluorescence photomicrograph (a photocopy of which isshown in FIG. 17b) was taken with the PDMS microfluidic stamp still inplace on the tissue culture plate surface in order to show theoverlaying weaving channel structures. The color of each of theconfluent layers of cells as viewed by fluorescence microscopy, isindicated on the figure above each square pattern feature. The blurredred spots 1080, 1082 and the blurred green spot 1084 comprise cellslocated in the channel structure of the top level of the microfluidicstamp above the focal plane of the microscope.

[0210] After removing the microfluidic stamp from the surface of thetissue culture plate, the surface was placed in tissue culture medium,as previously described, and cultured, as previously described, to allowthe two cell types to grow and spread together. FIG. 17c shows a portionof the image of FIG. 17b illustrating a patterned feature comprisinggreen deposited ECV cells and red deposited BCE cells. The two regionscontaining cells are separated by an intermediate region of the tissueculture plate surface (set off by dotted white lines), which is free ofcells. FIG. 17d shows a photocopy of a fluorescence photomicrographtaken of the identical region of the tissue culture plate surface taken20 hours after removal of the stamp and subsequent culture of the plate.FIGS. 17c and 17 d are registered, and the dotted intermediate region ofFIG. 17d comprises the region in FIG. 17c that was initially cell free.As can be seen, after 20 hours of culture subsequent to removal of themicrofluidic stamp, both cell types have divided, grown, and spreadtogether within the region that was initially cell free. FIG. 17e showsthe same region as shown FIG. 17d, also after 20 hours of culturesubsequent to removing the stamp, except as viewed with phase contrastlight microscopy.

[0211] While several embodiments of the invention have been describedand illustrated herein, those of ordinary skill in the art will readilyenvision a variety of other means and structures for performing thefunctions and/or obtaing the results or advantages described herein, andeach of such variations or modifications is deemed to be within thescope of the present invention. More generally, those skilled in the artwould readily appreciate that all parameters, dimensions, materials, andconfigurations (list modified as appropriate) described herein are meantto be exemplary and that actual parameters, dimensions, materials, andconfigurations will depend upon specific applications for which theteachings of the present invention are used. Those skilled in the artwill recognize, or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described herein. It is, therefore, to be understood that theforegoing embodiments are presented by way of example only and that,within the scope of the appended claims and equivalents thereto, theinvention may be practiced otherwise than as specifically described. Thepresent invention is directed to each individual feature, system,material and/or method described herein. In addition, any combination oftwo or more such features, systems, materials and/or methods, providedthat such features, systems, materials and/or methods are not mutuallyinconsistent, is included within the scope of the present invention. Inthe claims, all transitional phrases or phrases of inclusion, such as“comprising,” “including,” “carrying,” “having,” “containing,” and thelike are to be understood to be open-ended, i.e. to mean “including butnot limited to.” Only the transitional phrases or phrases of inclusion“consisting of” and “consisting essentially of” are to be interpreted asclosed or semi-closed phrases, respectively, as set forth in MPEPsection 2111.03.

What is claimed:
 1. A microfluidic network comprising: a polymericstructure including therein at least a first and a secondnon-fluidically interconnected fluid flow paths, with at least the firstflow path comprising a series of interconnected channels within thepolymeric structure, the series of interconnected channels including atleast one first channel disposed within a first level of the structure,at least one second channel disposed within a second level of thestructure, and at least one connecting channel fluidicallyinterconnecting the first channel and the second channel, where at leastone channel within the structure has a cross-sectional dimension notexceeding about 500 μm, and where the structure includes at least onechannel disposed within the first level of the structure that isnon-parallel to at least one channel disposed within the second level ofthe structure.
 2. The microfluidic network as in claim 1, wherein eachof the first and second flow paths comprises a series of interconnectedchannels within the polymeric structure, and wherein each series ofinterconnected channels includes at least one first channel disposedwithin a first level of the structure, at least one second channeldisposed within a second level of the structure, and at least oneconnecting channel fluidically interconnecting the first channel and thesecond channel.
 3. The microfluidic network as in claim 1, wherein atleast one second channel of the first flow path that is disposed withinthe second level of the structure is non-parallel to at least one firstchannel of the first flow path that is disposed within the first levelof the structure.
 4. The microfluidic network as in claim 1, wherein, atleast one channel of the first fluid flow path crosses over at least onechannel of the second fluid flow path, such that a perpendicularprojection of the channel of the first flow path and a perpendicularprojection of the channel of the second flow path onto a surfacedefining at least one of the first and second level at least partiallyoverlap each other.
 5. The microfluidic network as in claim 1, whereinthe polymeric structure is formed of an elastomeric material.
 6. Themicrofluidic network as in claim 5, wherein the elastomeric materialcomprises a silicone polymer.
 7. The microfluidic network as in claim 6,wherein the silicone polymer comprises poly(dimethylsiloxane).
 8. Themicrofluidic network as in claim 1, wherein the structure is comprisedof at least one discrete layer of polymeric material.
 9. Themicrofluidic network as in claim 8, wherein the structure is comprisedof at least two discrete layers of polymeric material, each layerincluding at least one channel therein, the layers being stacked uponeach other.
 10. The microfluidic network as in claim 9, wherein a firstdiscrete layer of the structure includes a surface defining the firstlevel of the structure and having the at least one first channeldisposed therein and further includes at least one channel traversing athickness of the layer and forming the at least one connecting channel,and wherein a second discrete layer of the structure includes a surfacedefining the second level of the structure and having the at least onesecond channel disposed therein.
 11. The microfluidic network as inclaim 9, wherein the structure is comprised of at least three discretelayers of polymeric material, a first discrete layer of the structuredefining the first level of the structure and having the at least onefirst channel disposed therein, a second discrete layer of the structureincluding at least one channel traversing a thickness of the layer andforming the at least one connecting channel, and a third discrete layerof the structure defining the second level of the structure and havingthe at least one second channel disposed therein.
 12. The microfluidicnetwork as in claim 9, wherein each of the at least two discrete layersis in conformal contact with another of the discrete layers.
 13. Themicrofluidic network as in claim 9, wherein each of the at least twodiscrete layers is irreversibly sealed to another of the discretelayers.
 14. The microfluidic network as in claim 8, wherein the at leastone discrete layer comprises a polymeric membrane including a firstsurface defining the first level of the structure and having the atleast one first channel disposed therein, a second surface defining thesecond level of the structure and having the at least one second channeldisposed therein, and a polymeric region intermediate the first surfaceand the second surface, the region including the at least one connectingchannel therethrough fluidically interconnecting the first channeldisposed in the first surface and the second channel disposed in thesecond surface of the membrane.
 15. The microfluidic network as in claim14, wherein at least the first surface of the polymeric membrane is inconformal contact with a surface of a substrate.
 16. The microfluidicnetwork as in claim 15, wherein the first surface of the polymericmembrane is irreversibly sealed to the surface of the substrate.
 17. Themicrofluidic network as in claim 15, wherein the substrate is formedfrom the same material forming the polymeric membrane.
 18. Themicrofluidic network as in claim 15, wherein the surface of thesubstrate is essentially planar.
 19. The microfluidic network as inclaim 15, wherein the surface of the substrate is curved.
 20. Themicrofluidic network as in claim 15, wherein the first surface of thepolymeric membrane is in conformal contact with a surface of a firstsubstrate and the second surface of the polymeric membrane is inconformal contact with a surface of a second substrate.
 21. Themicrofluidic network as in claim 20, wherein the first and secondsubstrates are formed of different materials.
 22. The microfluidicnetwork as in claim 20, wherein the first and second substrates areformed of the same material.
 23. The microfluidic network as in claim22, wherein the material forming the first and second substrates is thesame as the material forming the polymeric membrane.
 24. Themicrofluidic network as in claim 20, wherein the first surface of thepolymeric membrane is irreversibly sealed to the surface of the firstsubstrate.
 25. The microfluidic network as in claim 24, wherein thesecond surface of the polymeric membrane is irreversibly sealed to thesurface of the second substrate.
 26. The microfluidic network as inclaim 14, wherein the microfluidic network comprises a plurality ofdiscrete layers comprising a plurality of polymeric membranes stackedone upon another.
 27. The microfluidic network as in claim 1, wherein atleast one channel within the structure has a cross-sectional dimensionnot exceeding about 250 μm.
 28. The microfluidic network as in claim 27,wherein at least one channel within the structure has a cross-sectionaldimension not exceeding about 100 μm.
 29. The microfluidic network as inclaim 28, wherein at least one channel within the structure has across-sectional dimension not exceeding about 50 μm.
 30. Themicrofluidic network as in claim 29, wherein at least one channel withinthe structure has a cross-sectional dimension not exceeding about 20 μm.31. A microfluidic network comprising: an elastomeric structureincluding therein at least a one fluid flow path, with the flow pathcomprising a series of interconnected channels within the structure, theseries of interconnected channels including at least one first channeldisposed within a first level of the structure, at least one secondchannel disposed within a second level of the structure, and at leastone connecting channel fluidically interconnecting the first channel andthe second channel, where at least one channel within the structure hascross-sectional dimension not exceeding about 500 μm, where thestructure includes at least one channel disposed within the first levelof the structure that is non-parallel to at least one channel disposedwithin the second level of the structure; wherein the structure iscomprised of at least two discrete layers of polymeric material, eachlayer including at least one channel therein, the layers being stackedone upon each other; wherein a first discrete layer of the structureincludes a surface defining the first level of the structure and havingthe at least one first channel disposed therein and further includes atleast one channel traversing a thickness of the layer and forming the atleast one connecting channel; and wherein a second discrete layer of thestructure includes a surface defining the second level of the structureand having the at least one second channel disposed therein.
 32. Amicrofluidic network comprising: an elastomeric structure includingtherein at least a one fluid flow path, with the flow path comprising aseries of interconnected channels within the structure, the series ofinterconnected channels including at least one first channel disposedwithin a first level of the structure, at least one second channeldisposed within a second level of the structure, and at least oneconnecting channel fluidically interconnecting the first channel and thesecond channel, where at least one channel within the structure hascross-sectional dimension not exceeding about 500 μm, and where thestructure includes at least one channel disposed within the first levelof the structure that is non-parallel to at least one channel disposedwithin the second level of the structure; wherein the structure iscomprised of at least one discrete layer of polymeric material; whereinthe at least one discrete layer comprises a polymeric membrane includinga first surface defining the first level of the structure and having theat least one first channel disposed therein, a second surface definingthe second level of the structure and having the at least one secondchannel disposed therein, and a polymeric region intermediate the firstsurface and the second surface, the region including the at least oneconnecting channel therethrough fluidically interconnecting the firstchannel disposed in the first surface and the second channel disposed inthe second surface of the membrane.
 33. The microfluidic network as inclaim 31 or 32 wherein at least one channel within the structure has across-sectional dimension not exceeding about 250 μm.
 34. Themicrofluidic network as in claim 33, wherein at least one channel withinthe structure has a cross-sectional dimension not exceeding about 100μm.
 35. The microfluidic network as in claim 34, wherein at least onechannel within the structure has a cross-sectional dimension notexceeding about 50 μm.
 36. The microfluidic network as in claim 35,wherein at least one channel within the structure has a cross-sectionaldimension not exceeding about 20 μm.
 37. The microfluidic network as inclaim 32, wherein the structure comprises a plurality of the polymericmembranes stacked upon each other.
 38. A polymeric membrane comprising:a first surface including at least one channel disposed therein; asecond surface including at least one channel disposed therein; and apolymeric region intermediate the first surface and the second surface,the region including at least one connecting channel therethroughfluidically interconnecting the channel disposed in the first surfaceand the channel disposed in the second surface of the membrane, where atleast one channel has a cross-sectional dimension not exceeding about500 μm.
 39. The polymeric membrane as in claim 38, wherein at least thefirst surface of the polymeric membrane is in conformal contact with asurface of a substrate.
 40. The polymeric membrane as in claim 39,wherein the first surface of the polymeric membrane is irreversiblysealed to the surface of the substrate.
 41. The polymeric membrane as inclaim 39, wherein the substrate is formed from the same material formingthe polymeric membrane.
 42. The polymeric membrane as in claim 39,wherein the surface of the substrate is essentially planar.
 43. Thepolymeric membrane as in claim 39, wherein the surface of the substrateis includes at least one topological feature thereon.
 44. The polymericmembrane as in claim 43, wherein the at least one topological feature onthe surface of the substrate is shaped to be matable with a topologicalfeature on the first surface of the polymeric membrane.
 45. Thepolymeric membrane as in claim 39, wherein the first surface of thepolymeric membrane is in conformal contact with a surface of a firstsubstrate and the second surface of the polymeric membrane is inconformal contact with a surface of a second substrate.
 46. Thepolymeric membrane as in claim 45, wherein the first and secondsubstrates are formed of different materials.
 47. The polymeric membraneas in claim 45, wherein the first and second substrates are formed ofthe same material.
 48. The polymeric membrane as in claim 47, whereinthe material forming the first and second substrates is the same as thematerial forming the polymeric membrane.
 49. The polymeric membrane asin claim 38, wherein the polymeric membrane forms one of a plurality ofdiscrete layers stacked one upon another.
 50. The polymeric membrane asin claim 38, wherein at least one channel within the membrane has across-sectional dimension not exceeding about 250 μm.
 51. The polymericmembrane as in claim 50, wherein at least one channel within themembrane has a cross-sectional dimension not exceeding about 100 μm. 52.The polymeric membrane as in claim 51, wherein at least one channelwithin the membrane has a cross-sectional dimension not exceeding about50 μm.
 53. The polymeric membrane as in claim 52, wherein at least onechannel within the membrane has a cross-sectional dimension notexceeding about 20 μm.
 54. A method for forming a microfluidic networkstructure comprising: providing at least one mold substrate; forming atleast one topological feature on a surface of the mold substrate to forma first mold master, where at least one of said at least one topologicalfeature is a two-level topological feature characterized by a firstportion having a first depth or height with respect to a region of thesurface adjacent to the feature and a second portion, integrallyconnected to the first portion, having a second depth or height withrespect to the region of the surface adjacent to the feature, which isgreater than the first depth or height; contacting the surface with afirst hardenable liquid; hardening the liquid thereby creating a firstmolded replica of the surface; removing the first molded replica fromthe first mold master; and assembling the first molded replica into astructure comprising a microfluidic network having at least a one fluidflow path comprising a series of interconnected channels within thestructure, the series of interconnected channels including at least onefirst channel disposed within a first level of the structure, at leastone second channel disposed within a second level of the structure, andat least one connecting channel fluidically interconnecting the firstchannel and the second channel, where at least one of which channels hasa cross-sectional dimension not exceeding about 500 μm and where thestructure includes at least one channel disposed within the first levelof the structure that is non-parallel to at least one channel disposedwithin the second level of the structure.
 55. The method for forming amicrofluidic network structure as in claim 54, wherein the topologicalfeatures comprise protrusions from the surface of the first mold master,and wherein the first portion of the at least one two-level topologicalfeature has a first height with respect to the region of the surfaceadjacent to the two-level topological feature and the second portion hasa second height with respect to the region of the surface adjacent tothe feature, which is greater than the first height.
 56. The method forforming a microfluidic network structure as in claim 55, wherein thefirst molded replica formed by the hardening step and removing stepincludes a first surface forming the first level of the microfluidicnetwork formed in the assembling step, and wherein the at least onefirst channel disposed in the first level is molded by the first portionof the at least one two-level topological feature.
 57. The method forforming a microfluidic network structure as in claim 56, wherein thefirst molded replica formed by the hardening step and removing stepfurther includes the at least one connecting channel of the microfluidicnetwork formed in the assembling step, and wherein the connectingchannel is molded, at least in part, by the second portion of the atleast one two-level topological feature.
 58. The method for forming amicrofluidic network structure as in claim 55, wherein the contactingstep comprises: creating a layer of the first hardenable liquid on thesurface of the first mold master, the layer having a depth exceeding thefirst height but not exceeding the second height.
 59. The method forforming a microfluidic network structure as in claim 58, wherein thecreating step further comprises the step of: bringing a surface of asecond mold substrate into contact with a surface of the second portionof the at least one two-level topological feature in the surface of thefirst mold master.
 60. The method for forming a microfluidic networkstructure as in claim 59, wherein the surface of the second moldsubstrate is an essentially planar, featureless surface.
 61. The methodfor forming a microfluidic network structure as in claim 59, wherein thesecond mold substrate comprises a second mold master and wherein thesurface of the second mold master includes at least one topologicalfeature formed thereon.
 62. The method for forming a microfluidicnetwork structure as in claim 61, wherein at least one topologicalfeature in the surface of the second mold master is formed by aphotolithography process.
 63. The method for forming a microfluidicnetwork structure as in claim 61, wherein the surface of the second moldmaster comprises a molded replica of another surface including at leastone topological feature thereon.
 64. The method for forming amicrofluidic network structure as in claim 63, wherein the second moldmaster is formed from an elastomeric material.
 65. The method forforming a microfluidic network structure as in claim 61, wherein the atleast one topological feature in the surface of the second mold mastercomprises a protrusion from the surface.
 66. The method for forming amicrofluidic network structure as in claim 65, wherein the first moldedreplica formed by the hardening step and removing step includes a secondsurface forming the second level of the microfluidic network formed inthe assembling step, and wherein the at least one second channeldisposed in the second level is molded by the at least one topologicalfeature in the surface of the second mold master.
 67. The method forforming a microfluidic network structure as in claim 65, wherein thesurface of the second mold master includes at least one two-leveltopological feature thereon, which two-level topological feature ischaracterized by a first portion having a first height with respect to aregion of the surface adjacent to the feature and a second portion,integrally connected to the first portion, having a second height withrespect to the region of the surface adjacent to the feature, which isgreater than the first height.
 68. The method for forming a microfluidicnetwork structure as in claim 67, wherein at least a portion of thesecond portion of the two-level topological feature of the second moldmaster is shaped and positioned to mate with at least a portion of thesecond portion of a two-level topological feature of the first moldmaster when the mold masters are brought together in the bringing step.69. The method for forming a microfluidic network structure as in claim68, wherein at least a portion of the second portion of the two-leveltopological feature of the second mold master is shaped and positionedto interdigitate with at least a portion of the second portion of atwo-level topological feature of the first mold master when the moldmasters are brought together in the bringing step.
 70. The method forforming a microfluidic network structure as in claim 68, wherein thefirst molded replica formed during the hardening step and removing stepfurther includes the at least one connecting channel of the microfluidicnetwork formed in the assembling step, and wherein the connectingchannel is molded, at least in part, by the second portion of the atleast one two-level topological feature of the second mold master. 71.The method for forming a microfluidic network structure as in claim 61,wherein at least one topological feature on the surface of the firstmold master comprises a first alignment element and wherein at least onetopological feature on the second mold master comprises a secondalignment element, the second alignment element shaped to be matablewith the first alignment element.
 72. The method for forming amicrofluidic network structure as in claim 71, wherein both of the firstand second alignment elements comprise topological features that do notmold, during the contacting and hardening steps, channels in fluidcommunication with the at least one fluid flow path in the microfluidicnetwork structure.
 73. The method for forming a microfluidic networkstructure as in claim 71, wherein the first and second alignmentelements comprise topological features that mate together during thebringing step, and wherein at least a portion of at least one connectingchannel of the microfluidic network structure is molded, at least inpart, from at least a portion of the mated topological features.
 74. Themethod for forming a microfluidic network structure as in claim 61,wherein the contacting step comprises: bringing the surface of the firstmold master into at least partial contact with the surface of the secondmold master; aligning the at least one topological feature of the firstmold master and the at least one topological feature of the second moldmaster with respect to each other to yield a desired alignment offeatures; applying the first hardenable liquid in contact with aperiphery of the interface between the first and second mold masters;and allowing the first hardenable liquid to flow into intersticesbetween the first and the second mold masters by capillary action. 75.The method for forming a microfluidic network structure as in claim 61,wherein the contacting step comprises: forming a layer of the firsthardenable liquid on the surface of the first mold master; bringing thesurface of the second mold master into at least partial contact with thesurface of the first mold master; and aligning the at least onetopological feature of the first mold master and the at least onetopological feature of the second mold master with respect to each otherto yield a desired alignment of features.
 76. The method for forming amicrofluidic network structure as in claim 74 or 75, further comprising:interdigitating at least a portion of the at least one topologicalfeature of the first mold master and at least a portion of the at leastone topological feature of the second mold master.
 77. The method forforming a microfluidic network structure as in claim 61, wherein theremoving step comprises: applying a force to at least one of the firstand the second mold masters tending to separate the masters from eachother; removing the first molded replica from the surface of the firstmold master while leaving the first molded replica in contact with andsupported by the surface of the second mold master; and removing thesecond mold master from the first molded replica.
 78. The method forforming a microfluidic network structure as in claim 77, furthercomprising after the step of removing the first molded replica from thesurface of the first mold master while leaving the first molded replicain contact with and supported by the surface of the second mold master,and before the step of removing the second mold master from the firstmolded replica, the step of: contacting the first molded replica with asupport surface.
 79. The method for forming a microfluidic networkstructure as in claim 54, wherein the first hardenable liquid comprisesa liquid able to solidify to form a solid polymeric material.
 80. Themethod for forming a microfluidic network structure as in claim 79,wherein the first hardenable liquid comprises a curable prepolymer of anelastomeric polymer.
 81. The method for forming a microfluidic networkstructure as in claim 80, wherein the first hardenable liquid comprisesa curable prepolymer of poly(dimethylsiloxane).
 82. The method forforming a microfluidic network structure as in claim 79, wherein thehardening step comprises applying heat to the first hardenable liquid.83. The method for forming a microfluidic network structure as in claim79, wherein the hardening step comprises applying ultraviolet radiationto the first hardenable liquid.
 84. The method for forming amicrofluidic network structure as in claim 54, wherein the assemblingstep comprises: providing a first support substrate having at least oneoxidizable surface; oxidizing the oxidizable surface of the firstsupport substrate and the first molded replica; bringing the surface ofthe first support substrate into conformal contact with at least aportion of a first surface of the first molded replica; and sealing thefirst molded replica to the first support substrate via chemicalreaction between the surfaces.
 85. The method for forming a microfluidicnetwork structure as in claim 84, wherein the oxidizable surface of thefirst support substrate is essentially planar having essentially nofeatures disposed thereon.
 86. The method for forming a microfluidicnetwork structure as in claim 85, wherein the first support substrate isformed of a different material than the material forming the firstmolded replica.
 87. The method for forming a microfluidic networkstructure as in claim 85, wherein the first support substrate is formedof a material that is the same as that forming the first molded replica.88. The method for forming a microfluidic network structure as in claim84, wherein the first support substrate comprises a second moldedreplica.
 89. The method for forming a microfluidic network structure asin claim 88, further comprising before the oxidizing step the steps of:bringing at least a portion of the first surface of the first moldedreplica into contact with at least a portion of a surface of the secondmolded replica; aligning molded features of the first molded replicawith molded features of the second molded replica to yield a desiredalignment of features; and separating the surfaces of the first moldedreplica and the second molded replica from each other without disruptingthe desired alignment of features.
 90. The method for forming amicrofluidic network structure as in claim 88, further comprising afterthe oxidizing step and before the bringing step the steps of: placing aliquid that is essentially non-reactive with the surfaces oxidized inthe oxidizing step in contact with at least one of the surfaces oxidizedin the oxidizing step; disposing the first surface of the first moldedreplica and a surface of the second molded replica adjacent to eachother such that they are separated from each other by a continuous layerof the liquid that is essentially non-reactive with the surfacesoxidized in the oxidizing step; aligning molded features of the firstmolded replica with molded features of the second molded replica toyield a desired alignment of features; and removing the liquid that isessentially non-reactive with the surfaces oxidized in the oxidizingstep from between the surfaces.
 91. The method for forming amicrofluidic network structure as in claim 90, wherein the stepcomprising removing the liquid that is essentially non-reactive with thesurfaces oxidized in the oxidizing step from between the surfaces andthe bringing step comprise a single step.
 92. The method for forming amicrofluidic network structure as in claim 90, wherein the liquid thatis essentially non-reactive with the surfaces oxidized in the oxidizingstep is removed from between the surfaces by evaporation.
 93. The methodfor forming a microfluidic network structure as in claim 84, wherein theassembling step further comprises: providing a second support substratehaving at least one oxidizable surface; oxidizing the oxidizable surfaceof the second support substrate and the first molded replica; bringingthe surface of the second support substrate into conformal contact withat least a portion of a second surface of the first molded replica; andsealing the first molded replica to the second support substrate viachemical reaction between the surfaces.
 94. The method for forming amicrofluidic network structure as in claim 93, wherein the oxidizablesurface of the second support substrate is essentially planar havingessentially no features disposed thereon.
 95. The method for forming amicrofluidic network structure as in claim 94, wherein the secondsupport substrate is formed of a different material than the materialforming the first molded replica.
 96. The method for forming amicrofluidic network structure as in claim 94, wherein the secondsupport substrate is formed of a material that is the same as thatforming the first molded replica.
 97. The method for forming amicrofluidic network structure as in claim 93, wherein the secondsupport substrate comprises a second molded replica.
 98. The method forforming a microfluidic network structure as in claim 54, furthercomprising after the assembling step the steps of: at least partiallyfilling the at least one fluid flow path of the microfluidic networkwith a second hardenable liquid; solidifying the second hardenableliquid into a molded article having a structure conforming to the flowpath of the microfluidic network; and removing the microfluidic networkstructure surrounding the molded article.
 99. A method for forming amolded structure comprising: providing at least one mold substrate;forming at least one two-level topological feature having at least onecross-sectional dimension not exceeding about 500 μm on a surface of thesubstrate to form a mold master, which two-level topological feature ischaracterized by a first portion having a first depth or height withrespect to a region of the surface adjacent to the feature and a secondportion, integrally connected to the first portion, having a seconddepth or height with respect to the region of the surface adjacent tothe feature, which is greater than the first depth or height; contactingthe surface with a hardenable liquid; hardening the liquid therebycreating a molded replica of the surface; and removing the moldedreplica from the mold master.
 100. The method for forming a moldedstructure as in claim 99, wherein the molded replica formed by thehardening step and removing step includes a first surface with at leastone channel disposed therein that is molded by the first portion of theat least one two-level topological feature and further includes at leastone connecting channel fluidically interconnected to and orientedessentially perpendicularly to the channel disposed in the first surfaceof the molded replica, which connecting channel is molded by the secondportion of the two-level topological feature.
 101. The method forforming a molded structure as in claim 99, wherein the mold substratecomprises a silicon wafer.
 102. The method for forming a moldedstructure as in claim 101, wherein at least one surface of the siliconwafer is coated with at least a first layer of photoresist having asurface forming a surface of the substrate on which the at least onetopological feature is formed in the forming step.
 103. The method forforming a molded structure as in claim 102, wherein the photoresistcomprises a positive photoresist.
 104. The method for forming a moldedstructure as in claim 102, wherein the photoresist comprises a negativephotoresist.
 105. The method for forming a molded structure as in claim102, wherein the forming step comprises: providing a first photo maskdefining a first pattern; exposing the surface of the first layer ofphotoresist to radiation through the first photo mask; coating thesurface of the first layer of photoresist with a second layer ofphotoresist; providing a second photo mask defining a second pattern;and exposing a surface of the second layer of photoresist to radiationthrough the second photo mask.
 106. The method for forming a moldedstructure as in claim 105, wherein the first and second photomaskscomprise printed transparencies.
 107. The method for forming a moldedstructure as in claim 106, wherein the first and second patterns aredesigned by a computer assisted design program and are printed onto thetransparencies with a high resolution printer.
 108. The method forforming a molded structure as in claim 105, further comprising aftereach of the exposing steps, the step of: developing the photoresistlayer with a developing agent that selectively removes photoresistmaterial based on whether the photoresist material has been exposed toradiation through the photomask to yield a positive relief pattern inphotoresist with topological features corresponding to the pattern ofthe photo mask.
 109. The method for forming a molded structure as inclaim 105, further comprising after the second exposing step, the stepof: developing the first and second photoresist layers with a developingagent that selectively removes photoresist material based on whether thephotoresist material has been exposed to radiation through either of thefirst or second photomasks to yield a positive relief pattern inphotoresist with topological features corresponding to the first andsecond patterns of the first and second photo masks.
 110. The method forforming a molded structure as in claim 105, further comprising after thestep for providing the second photo mask and before the second exposingstep, the step of: aligning the second photo mask so that the secondpattern has a desired orientation and position with respect to a priororientation and position of the first pattern of the first photo mask.111. The method for forming a molded structure as in claim 110, whereinfeatures of the first pattern of the first photo mask correspond tofirst portions of the at least one two-level topological feature andwherein features of the second pattern of the second photo maskcorrespond to second portions of the at least one two-level topologicalfeature.
 112. A method for forming topological features on a surface ofa material comprising: exposing portions of surface of a first layer ofphotoresist to radiation in a first pattern; coating the surface of thefirst layer of photoresist with a second layer of photoresist; exposingportions of a surface of the second layer of photoresist to radiation ina second pattern different from the first pattern; and developing thefirst and second photoresist layers with a developing agent to yield apositive relief pattern in photoresist, the positive relief patternincluding at least one two-level topological feature having at least onecross-sectional dimension not exceeding about 500 μm, which two-leveltopological feature is characterized by a first portion having a firstheight with respect to the surface of the material and a second portion,integrally connected to the first portion, having a second height withrespect to the surface of the material.
 113. A method for forming amolded structure comprising: providing a first mold master having asurface formed of an elastomeric material and including at least onetopological feature with at least one cross-sectional dimension notexceeding about 500 μm thereon; providing a second mold master having asurface including at least one topological feature with at least onecross-sectional dimension not exceeding about 500 μm thereon; placing ahardenable liquid in contact with the surface of at least one of thefirst and second mold master; bringing the surface of the first moldmaster into at least partial contact with the surface of the second moldmaster; hardening the liquid thereby creating a molded replica of thesurface of the first mold master and the surface of the second moldmaster; and removing the molded replica from at least one of the moldmasters.
 114. A method for forming a molded structure comprising:providing a first mold master having a surface including at least afirst topological feature with at least one cross-sectional dimensionnot exceeding about 500 μm thereon and at least a second topologicalfeature comprising a first alignment element; providing a second moldmaster having a surface including at least a first topological featurewith at least one cross-sectional dimension not exceeding about 500 μmthereon and at least a second topological feature comprising a secondalignment element having a shape that is matable to the shape of thefirst alignment element; placing a hardenable liquid in contact with thesurface of at least one of the first and second mold master; bringingthe surface of the first mold master into at least partial contact withthe surface of the second mold master; aligning the first topologicalfeatures of the first and second mold masters with respect to each otherby adjusting a position of the first mold master with respect to aposition of the second mold master until the first alignment elementmatingly engages the second alignment element; hardening the liquidthereby creating a molded replica of the surface of the first moldmaster and the surface of the second mold master; and removing themolded replica from at least one of the mold masters.
 115. The methodfor forming a microfluidic network structure as in claim 114, whereinboth of the first and second alignment elements comprise topologicalfeatures that do not mold, during the hardening step, any features ofthe final molded structure.
 116. The method for forming a microfluidicnetwork structure as in claim 114, wherein the first and secondalignment elements comprise topological features that together mold,during the hardening step, at least a portion of at least one feature ofthe final molded structure.
 117. The method for forming a moldedstructure as in claim 114, wherein at least one of the first mold masterand the second mold master is formed of an elastomeric material.
 118. Amethod for aligning and sealing together surfaces comprising: disposingtwo surfaces comprised of different materials, at least one of whichsurfaces is oxidized, adjacent to each other such that they areseparated from each other by a continuous layer of a liquid that isessentially non-reactive with the surfaces; aligning the surfaces withrespect to each other; and removing the liquid from between thesurfaces, thereby sealing the surfaces together via a chemical reactionbetween the surfaces.
 119. The method for aligning and sealing togethersurfaces as in claim 118, wherein the at least one surface that isoxidized is oxidized by exposing the surface to an oxygen-containingplasma.
 120. The method for aligning and sealing together surfaces as inclaim 118, wherein in the removing step, the liquid that is essentiallynon-reactive with the surfaces is removed by evaporation.
 121. Themethod for aligning and sealing together surfaces as in claim 118,wherein the two surfaces are selected from the group of materialsconsisting of: silicone polymers; glass; silicon; silicon oxide; quartz;silicon nitride; polyethylene; polystyrene; epoxy polymers; and glassycarbon.
 122. The method for aligning and sealing together surfaces as inclaim 121, wherein at least one of the two surfaces comprises a siliconepolymer.
 123. The method for aligning and sealing together surfaces asin claim 122, wherein the silicone polymer comprisespoly(dimethylsiloxane).
 124. The method for aligning and sealingtogether surfaces as in claim 118, wherein the liquid that isessentially non-reactive with the surfaces is selected from the groupconsisting of water, alcohols, and mixtures thereof.
 125. The method foraligning and sealing together surfaces as in claim 124, wherein theliquid that is essentially non-reactive with the surfaces includesmethonol.
 126. The method for aligning and sealing together surfaces asin claim 124, wherein the liquid that is essentially non-reactive withthe surfaces includes trifluoroethanol.
 127. The method for aligning andsealing together surfaces as in claim 118, wherein at least one of thetwo surfaces includes at least one self-alignment element thereon andwherein in the aligning step, the surfaces self align with respect toeach other, the self-alignment driven by the surface tension of theliquid that is essentially non-reactive with the surfaces and a shapeand position of the self alignment element.
 128. A method for molding anarticle comprising: providing a first mold master having a surface witha first set of surface properties; providing a second mold master havinga surface with a second set of surface properties, wherein the surfaceof at least the first mold master is formed of an elastomeric material,and wherein at least one of the first and second mold master has asurface including at least one topological feature with at least onecross-sectional dimension not exceeding about 500 μm thereon; placing ahardenable liquid in contact with the surface of at least one of thefirst and second mold master; bringing the surface of the first moldmaster into at least partial contact with the surface of the second moldmaster; hardening the liquid thereby creating a molded replica of thesurface of the first mold master and the surface of the second moldmaster; separating the masters from each other; and removing the moldedreplica from the surface of the first mold master while leaving themolded replica in contact with and supported by the surface of thesecond mold master.
 129. The method for molding an article as in claim128, wherein the separating step comprises applying a peeling force toat least one of the first and second mold masters.
 130. The method formolding an article as in claim 128, wherein the surface of at least oneof the first and second mold masters has been silanized.
 131. The methodfor molding an article as in claim 128, wherein the elastomeric materialcomprises a silicone polymer.
 132. The method for molding an article asin claim 131, wherein the silicone polymer comprisespoly(dimethylsiloxane).
 133. The method for molding an article as inclaim 132, wherein the molded replica is formed ofpoly(dimethylsiloxane).
 134. The method for molding an article as inclaim 133, wherein the surface of the second mold master is formed of amaterial other than poly(dimethylsiloxane).
 135. A microfluidic networkcomprising: a polymeric structure including therein at least a first anda second non-fluidically-interconnected fluid flow paths, the first flowpath comprising at least two non-colinear, interconnected channelsdisposed within a first plane and the second flow path comprising atleast one channel disposed within a second plane that is non-parallelwith the first plane, and where at least one channel within thestructure has a cross-sectional dimension not exceeding about 500 μm.136. The microfluidic network as in claim 135, wherein the second flowpath comprises at least two non-colinear, interconnected channelsdefining the second plane.
 137. The microfluidic network as in claim135, wherein at least one of the first and second flow paths comprisesat least a first, a second, and a third interconnected channels, thefirst and second channels defining together a plane intersected by thethird channel.